Pegylated OXM variants

ABSTRACT

A composition which includes oxyntomodulin and polyethylene glycol polymer (PEG polymer) linked via a reversible linker such as 9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) is disclosed. Pharmaceutical compositions comprising the reverse pegylated oxyntomodulin and methods of using same are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase Application of PCT InternationalApplication No. PCT/IL2013/050481, International Filing Date Jun. 4,2013, claiming priority to U.S. Provisional Patent Application No.61/655,367, filed Jun. 4, 2012, both of which are incorporated byreference herein in their entirety.

FIELD OF INVENTION

A composition which includes oxyntomodulin and polyethylene glycolpolymer (PEG polymer) linked via a reversible linker such as9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS) is disclosed. Pharmaceutical compositions comprising the reversepegylated oxyntomodulin and methods of using same are also disclosed.

BACKGROUND OF THE INVENTION

The gastrointestinal tract is responsible on synthesize and releasing ofmany peptide hormones that regulate eating behavior including pancreaticprotein (PP), glucagon-like peptide 1 (GLP-1), peptide YY (PYY) andOxyntomodulin (OXM). OXM arises from a tissue-specific post-transitionalprocessing of proglucagon in the intestine and the CNS. It contains 37amino acids, including the complete glucagon sequence with a C-terminalbasic octapeptide extension that was shown to contribute to theproperties of OXM both in-vitro and in-vivo but was not alone sufficientfor the effects of the peptide. In response to food ingestion, OXM issecreted by intestinal L cells into the bloodstream proportionally tothe meal caloric content.

OXM enhances glucose clearance via stimulation of insulin secretionafter both oral and intraperitoneal administration. It also regulatesthe control of food intake. Intracerebroventricular (ICV) andintranuclear injection of OXM into the paraventricular and arcuatenuclei (ARC) of the hypothalamus inhibits re-feeding in fasting rats.This inhibition has also been demonstrated in freely fed rats at thestart of the dark phase. Moreover, peripheral administration of OXMdose-dependently inhibited both fast-induced and dark-phase food intake.

Unfavorable pharmacokinetics, such as a short serum half-life, canprevent the pharmaceutical development of many otherwise promising drugcandidates. Serum half-life is an empirical characteristic of amolecule, and must be determined experimentally for each new potentialdrug. For example, with lower molecular weight protein drugs,physiological clearance mechanisms such as renal filtration can make themaintenance of therapeutic levels of a drug unfeasible because of costor frequency of the required dosing regimen.

Proteins and especially short peptides are susceptible to denaturationor enzymatic degradation in the blood, liver or kidney. Accordingly,proteins typically have short circulatory half-lives of several hours.Because of their low stability, peptide drugs are usually delivered in asustained frequency so as to maintain an effective plasma concentrationof the active peptide. Moreover, since peptide drugs are usuallyadministered by infusion, frequent injection of peptide drugs causeconsiderable discomfort to a subject. Thus, there is a need fortechnologies that will prolong the half-lives of therapeutic proteinsand peptides while maintaining a high pharmacological efficacy thereof.Such desired peptide drugs should also meet the requirements of enhancedserum stability, high activity and a low probability of inducing anundesired immune response when injected into a subject.

The present invention relates to OXM derivative in which the half-lifeof the peptide is prolonged utilizing a reversible pegylationtechnology.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a composition consisting ofan oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to the amino terminus ofsaid oxyntomodulin via Fmoc or FMS.

In one embodiment, the invention relates to a composition consisting ofan oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to a lysine residue onposition number twelve (Lys₁₂) of said oxyntomodulin's amino acidsequence via Fmoc or FMS.

In another embodiment, the invention relates to a composition consistingof an oxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein said PEG polymer is attached to a lysine residue onposition number thirty (Lys₃₀) of said oxyntomodulin's amino acidsequence via Fmoc or FMS.

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 or to the Lysine residue on positionnumber 30 or to the amino terminus of said oxyntomodulin's amino acidsequence via 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method of reducing thedosing frequency of oxyntomodulin, consisting of the step of conjugatinga polyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 or to the Lysine residue on position number 30 or tothe amino terminus of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 12 or to a Lysine residue on position number 30 or to the aminoterminus of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

Other features and advantages of the present invention will becomeapparent from the following detailed description examples and figures.It should be understood, however, that the detailed description and thespecific examples while indicating preferred embodiments of theinvention are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows different variants of the PEG-FMS-OXM conjugate produced.

FIG. 2 is a graph showing the in vitro activity (cAMP quantitation) ofthe heterogeneous PEG₃₀-FMS-OXM and the three PEG₃₀-FMS-OXM variants(amino, Lys12 and Lys30) when incubated with CHO-K1 cellsover-expressing GLP-1 receptor.

FIG. 3 is a graph showing the in vivo activity of the heterogeneousPEG₃₀-FMS-OXM and the three PEG₃₀-FMS-OXM variants (amino, Lys12 andLys30) in the IPGTT model. All the compounds induced glucose tolerancecompared to vehicle group.

FIG. 4 shows the effect of the heterogeneous PEG30-FMS-OXM and the threePEG30-FMS-OXM variants (amino, Lys12 and Lys30) on body weight in maleob/ob mice.

FIG. 5 shows the effect of the heterogeneous PEG30-FMS-OXM and the threePEG30-FMS-OXM variants (amino, Lys12 and Lys30) on food intake in maleob/ob mice.

FIG. 6 shows the effect of the heterogeneous PEG30-FMS-OXM and the threePEG30-FMS-OXM variants (amino, Lys12 and Lys30) on non-fasting andfasting glucose in male ob/ob mice.

FIG. 7 shows the effect of MOD-6031, OXM and liraglutide on cumulativefood intake in male ob/ob mice.

FIG. 8 shows the effect of MOD-6031, OXM and liraglutide on body weightin male ob/ob mice.

FIG. 9 shows the effect of MOD-6031, OXM and liraglutide on freelyfeeding and fasted plasma glucose in male ob/ob mice.

FIG. 10 shows the effect of MOD-6031 and pair fed group on glucosetolerance (2 g/kg po) on day 2 of the study, in male ob/ob mice.

FIG. 11 shows the effect of MOD-6031 and pair fed group on glucosetolerance (2 g/kg po) on day 30 of the study, in male ob/ob mice

FIG. 12 shows the effect of MOD-6031, OXM and liraglutide on terminalplasma cholesterol in male ob/ob mice

DETAILED DESCRIPTION OF THE INVENTION

Provided herein is a long-acting oxyntomodulin and methods of producingand using same. In one aspect, the invention provides a compositioncomprising or consisting of a dual GLP-1/Glucagon receptor agonist, apolyethylene glycol polymer (PEG polymer) and 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS). In anotherembodiment, the invention provides a composition comprising orconsisting of an oxyntomodulin, a polyethylene glycol polymer (PEGpolymer) and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS). In another embodiment, the PEGpolymer is attached to a lysine residue on position number twelve(Lys₁₂) of the oxyntomodulin's amino acid sequence via Fmoc or FMS. Inone embodiment, a long-acting oxyntomodulin is a composition comprisingor consisting of oxyntomodulin and polyethylene glycol polymer (PEGpolymer) attached to a lysine residue on position number twelve (Lys₁₂)of the oxyntomodulin's amino acid sequence via Fmoc or FMS.

In another aspect, provided herein is a novel method for extending theserum half-life of peptides. This method is based on the reversibleattachment of a polyethylene glycol (PEG) chain to the peptide through achemical linker (called FMS or Fmoc) resulting in the slow release ofthe native peptide into the bloodstream. The released peptide can thenalso cross the blood brain barrier to enter the central nervous system(CNS) or any other target organ. In one embodiment, the unique chemicalstructure of the FMS linker leads to a specific rate of peptide release.

Hence, in another embodiment, provided herein is a method for extendingthe biological half-life of an OXM peptide. In another embodiment,provided herein is a method for extending the circulating time in abiological fluid of OXM, wherein said circulating time is extended bythe slow release of the intact OXM peptide. In another embodiment,extending said biological half-life or said circulating time of said OXMpeptide allows said OXM to cross the blood brain barrier and target theCNS. It will be well appreciated by the skilled artisan that thebiological fluid may be blood, sera, cerebrospinal fluid (CSF), and thelike.

In one embodiment, upon administration of the PEGylated oxyntomodulincomposition of the present invention into a subject, the oxyntomodulinis released into a biological fluid in the subject as a result ofchemical hydrolysis of said FMS or said Fmoc linker from saidcomposition. In another embodiment, the released oxyntomodulin is intactand regains complete GLP-1 and glucagon receptor binding activity. Inanother embodiment, chemically hydrolyzing said FMS or said Fmoc extendsthe circulating time of said OXM peptide in said biological fluid. Inanother embodiment, extending the circulating time of said OXM allowssaid OXM to cross the blood brain barrier and target the CNS. In anotherembodiment, extending the circulating time of said OXM allows said OXMto cross the blood brain barrier and target the hypothalamus. In anotherembodiment, extending the circulating time of said OXM allows said OXMto cross the blood brain barrier and target the arcuate nucleus.

In one aspect, the amino variant of PEG30-FMS-OXM (designated MOD-6031)is a site directed conjugate comprising OXM and mPEG(30)-SH linkedthrough a bi-functional linker (FMS or Fmoc). In another embodiment, theOXM peptide is connected through its terminal amine of the N-terminusside which reacts with the N-succinimide ester (NHS) group on the linkerfrom one side while mPEG(30)-SH is connected to the maleimide moiety ofthe FMS linker by its thiol group (see Examples herein). The Lys12 andLys30 variants are conjugated to the FMS linker through their aminegroup of Lys residues. In one embodiment, the reversible-pegylationmethod is utilized herein to generate the long lasting oxyntomodulin(OXM) peptides provided herein (e.g. PEG30-FMS-OXM).

In one embodiment, the terms dual “GLP-1/Glucagon receptor agonist” and“agonist” are used interchangeably herein. In another embodiment, theterms also include any GLP-1/Glucagon receptor agonist known in the art.In another embodiment, the preferred agonist is oxyntomodulin or OXM ora functional variant thereof.

In one embodiment, the term “functional” refers to the ability of theagonist or OXM provided herein to have biological activity, whichinclude but is not limited to, reducing weight, increasing insulinsensitivity, reducing insulin resistance, increasing energy expenditureinducing glucose tolerance, inducing glycemic control, improvingcholesterol levels, etc., as further provided herein.

In one embodiment, the invention provides a composition comprising anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein the PEG polymer is attached to a lysine residue onposition number thirty (Lys₃₀) of said oxyntomodulin amino acid sequencevia Fmoc or FMS. In one embodiment, a long-acting oxyntomodulin is acomposition comprising or consisting of oxyntomodulin and polyethyleneglycol polymer (PEG polymer) attached to a lysine residue on positionnumber twelve (Lys₃₀) of the oxyntomodulin amino acid sequence via Fmocor FMS.

In one embodiment, the invention provides a composition consisting of anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein the PEG polymer is attached to a lysine residue onposition number thirty (Lys₃₀) of said oxyntomodulin's amino acidsequence via Fmoc or FMS. In one embodiment, a long-acting oxyntomodulinis a composition comprising or consisting of oxyntomodulin andpolyethylene glycol polymer (PEG polymer) attached to a lysine residueon position number twelve (Lys₃₀) of the oxyntomodulin's amino acidsequence via Fmoc or FMS.

In one embodiment, the invention provides a composition comprising anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and a9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein the PEG polymer is attached to the amino terminus of saidoxyntomodulin via Fmoc or FMS. In one embodiment, a long-actingoxyntomodulin is a composition comprising or consisting of oxyntomodulinand polyethylene glycol polymer (PEG polymer) attached to the aminoterminus of the oxyntomodulin's amino acid sequence via Fmoc or FMS.

In one embodiment, the invention provides composition consisting of anoxyntomodulin, a polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS), wherein the PEG polymer is attached to the amino terminus of saidoxyntomodulin via Fmoc or FMS. In one embodiment, a long-actingoxyntomodulin is a composition comprising or consisting of oxyntomodulinand polyethylene glycol polymer (PEG polymer) attached to the aminoterminus of the oxyntomodulin's amino acid sequence via Fmoc or FMS.

In another embodiment, the present invention provides a compositioncomprising an oxyntomodulin peptide, and a polyethylene glycol (PEG)polymer conjugated to the oxyntomodulin peptide's lysine amino acid onposition twelve (Lys12) or position 30 (Lys30) or on the amino terminusof the oxyntomodulin peptide via a 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) linker. In another embodiment,the present invention provides a modified oxyntomodulin peptideconsisting of an oxyntomodulin peptide, and a polyethylene glycol (PEG)polymer conjugated to the oxyntomodulin peptide's lysine amino acid onposition twelve (Lys12) or position 30 (Lys30) or on the amino terminusof the oxyntomodulin peptide via a 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) linker. In another embodiment,the compositions where PEG is attached to oxyntomodulin at Lys12, Lys30or at the amino terminus are respectively referred to as the “Lys12variant,” the “Lys30 variant” or the “amino variant,” of oxyntomodulin.In one embodiment, the term “amino variant” is synonymous with“N-terminal variant”, “N′ variant” or “N-terminus variant”. It is to beunderstood that a skilled artisan may be guided by the present inventionto readily insert lysine residues in a site-specific or random mannerthroughout the OXM sequence in order to attach a linker (Fmoc orFMS)/PEG conjugate provided herein at these lysine residues. In oneembodiment, variants where one or more lysine residues are located indifferent positions throughout the OXM sequence and are used forconjugating OXM to PEG and cleavable linker (e.g. FMS or Fmoc), are alsoencompassed in the present invention.

In one embodiment, the present invention provides a compositioncomprising an oxyntomodulin peptide, and a polyethylene glycol (PEG)polymer conjugated to the oxyntomodulin peptide's lysine amino acid onposition twelve (Lys12) and position 30 (Lys30) via a9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS) linker. In another embodiment, the present invention provides acomposition comprising an oxyntomodulin peptide, and a polyethyleneglycol (PEG) polymer conjugated to the oxyntomodulin peptide's lysineamino acid on position twelve (Lys12) and on the amino terminus via a9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS) linker. In another embodiment, the present invention provides acomposition comprising an oxyntomodulin peptide, and a polyethyleneglycol (PEG) polymer conjugated to the oxyntomodulin peptide's lysineamino acid on position thirty (Lys30) and on the amino terminus via a9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS) linker.

In another embodiment, a long-acting oxyntomodulin is a pegylatedoxyntomodulin. In another embodiment, a long-acting oxyntomodulin is areversed pegylated oxyntomodulin. In another embodiment, the phrases“long-acting oxyntomodulin,” “reversed pegylated oxyntomodulin,”“reversible PEGylated OXM,” or “a composition comprising or consistingof oxyntomodulin, polyethylene glycol polymer (PEG polymer) and9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS)” are used interchangeably. In another embodiment, a long-actingoxyntomodulin is OXM linked to PEG via Fmoc or FMS. In anotherembodiment, the long-acting OXM is linked to Fmoc or FMS via its Lys12residue, or its Lys30 residue or its amino (N′) terminus.

In one embodiment, a long-acting oxyntomodulin of the inventioncomprises a PEG polymer. In another embodiment, a long-actingoxyntomodulin of the invention comprises a PEG polymer conjugated to theamino terminus of an oxyntomodulin peptide via Fmoc or FMS. In anotherembodiment, a long-acting oxyntomodulin of the invention comprises a PEGpolymer conjugated via Fmoc or FMS to lysine residues 12 or 30 of theoxyntomodulin peptide. In another embodiment, a long-actingoxyntomodulin of the invention comprises a PEG polymer conjugated viaFmoc or FMS to both the amino terminus of an oxyntomodulin peptide andto lysine residues 12 and 30 of oxyntomodulin.

In another embodiment, a long-acting oxyntomodulin is a compositioncomprising or consisting of oxyntomodulin, polyethylene glycol polymer(PEG polymer) and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of1:0.2-10:0.2-10. In another embodiment, a long-acting oxyntomodulin is acomposition comprising or consisting of oxyntomodulin, polyethyleneglycol polymer (PEG polymer) and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of1:0.5-2:0.5-2. In another embodiment, a long-acting oxyntomodulin is acomposition comprising or consisting of oxyntomodulin, polyethyleneglycol polymer (PEG polymer) and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1. Inanother embodiment, a long-acting oxyntomodulin includes a PEG polymerconjugated to the amino terminus of oxyntomodulin via Fmoc or FMS. Inanother embodiment, the molar ratio of OXM-PEG- and linker is1:1:1-1:1:3.5. In another embodiment, the molar ratio is 1:1:1-1:1:10.0.In another embodiment, the higher ratio of linker allows for optimizedyield of the composition.

In another embodiment, a long-acting oxyntomodulin is linked to PEG viaa reversible linker such as, but not limited to, Fmoc and FMS. Inanother embodiment, Fmoc and FMS are sensitive to bases and areremovable under physiological conditions. In another embodiment, areversible linker is a linker that is sensitive to bases and isremovable under physiological conditions. In another embodiment, areversible linker is a linker that is sensitive to bases and isremovable under physiological conditions in the blood, plasma, or lymph.In another embodiment, a reversible linker is a linker that is sensitiveto bases and is removable under physiological conditions in a bodyfluid. In another embodiment, a reversible linker is a linker that isremovable in a body fluid having a basic pH. In another embodiment, alinker that is sensitive to bases is cleaved upon exposure to a basicenvironment thus releasing OXM from the linker and PEG. In anotherembodiment, a linker that is sensitive to temperature is cleaved uponexposure to specific temperature that allows for such cleavage to takeplace. In another embodiment, the temperature that enables cleavage ofthe linker is within the physiological range.

In another embodiment, a reverse pegylated oxyntomodulin is acomposition wherein OXM is linked to PEG via a reversible linker. Inanother embodiment, a reverse pegylated oxyntomodulin releases free OXMupon exposure to a basic environment. In another embodiment, a reversepegylated oxyntomodulin releases free OXM upon exposure to blood orplasma. In another embodiment, a long-acting oxyntomodulin comprises PEGand oxyntomodulin that are not linked directly to each other, as instandard pegylation procedures, but rather both residues are linked todifferent positions of Fmoc or FMS which are highly sensitive to basesand are removable under regular physiological conditions. In anotherembodiment, regular physiological conditions include a physiologicenvironment such as the blood or plasma.

In another embodiment, the structures and the processes of making Fmocand FMS are described in U.S. Pat. No. 7,585,837. The disclosure of U.S.Pat. No. 7,585,837 is hereby incorporated by reference in its entirety.

In another embodiment, reverse pegylation renders OXM a long-acting OXM.In another embodiment, long-acting oxyntomodulin is an oxyntomodulinwith an extended biological half-life. In another embodiment, reversepegylation provides protection against degradation of OXM. In anotherembodiment, reverse pegylation effects the C_(max) of OXM and reducesside effects associated with administration of the composition providedherein. In another embodiment, reverse pegylation extends the T_(max) ofOXM. In another embodiment, reverse pegylation extends the circulatoryhalf-live of OXM. In another embodiment, reverse pegylated OXM hasimproved bioavailability compared to non-modified OXM. In anotherembodiment, reverse pegylated OXM has improved biological activitycompared to non-modified OXM. In another embodiment, reverse pegylationenhances the potency of OXM.

In other embodiments, a reverse pegylated OXM is at least equivalent tothe non-modified OXM, in terms of biochemical measures. In otherembodiments, a reverse pegylated OXM is at least equivalent to thenon-modified OXM, in terms of pharmacological measures. In otherembodiments, a reverse pegylated OXM is at least equivalent to thenon-modified OXM, in terms of binding capacity (Kd). In otherembodiments, a reverse pegylated OXM is at least equivalent to thenon-modified OXM, in terms of absorption through the digestive system.In other embodiments, a reverse pegylated OXM is more stable duringabsorption through the digestive system than non-modified OXM.

In another embodiment, a reverse pegylated OXM exhibits improved bloodarea under the curve (AUC) levels compared to free OXM. In anotherembodiment, a reverse pegylated OXM exhibits improved biologicalactivity and blood area under the curve (AUC) levels compared to freeOXM. In another embodiment, a reverse pegylated OXM exhibits improvedblood retention time (t_(1/2)) compared to free OXM. In anotherembodiment, a reverse pegylated OXM exhibits improved biologicalactivity and blood retention time (t_(1/2)) compared to free OXM. Inanother embodiment, a reverse pegylated OXM exhibits improved bloodC_(max) levels compared to free OXM, where in another embodiment itresults in a slower release process that reduces side effects associatedwith administration of the reverse pegylated compositions providedherein. In another embodiment, a reverse pegylated OXM exhibits improvedbiological activity and blood C_(max) levels compared to free OXM. Inanother embodiment, provided herein a method of improving OXM's AUC,C_(max), t_(1/2), biological activity, or any combination thereofcomprising or consisting of the step of conjugating a polyethyleneglycol polymer (PEG polymer) to the amino terminus of free OXM via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, improvement of OXM's AUC, C_(max), t_(1/2),biological activity, or any combination thereof by conjugating apolyethylene glycol polymer (PEG polymer) to the amino terminus of freeOXM via 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) enables the reduction in dosingfrequency of OXM. In another embodiment, provided herein a method forreducing a dosing frequency of OXM, comprising or consisting of the stepof conjugating a polyethylene glycol polymer (PEG polymer) to the aminoterminus or lysine residues of OXM via 9-fluorenylmethoxycarbonyl (Fmoc)or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS). In another embodiment,reverse pegylation of OXM is advantageous in permitting lower dosages tobe used.

In another embodiment, OXM comprises the amino acid sequence of SEQ IDNO: 1. In another embodiment, OXM consists of the amino acid sequence ofSEQ ID NO: 1. In another embodiment, SEQ ID NO: 1 comprises or consistsof the following amino acid (AA) sequence:HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO: 1). In anotherembodiment, OXM comprises or consists of the amino acid sequencedepicted in CAS No. 62340-29-8.

In another embodiment, OXM is human OXM or any mammal OXM. In anotherembodiment, OXM is also referred to as glucagon-37 or bioactiveenteroglucagon. In another embodiment, OXM is a dual GLP-1/Glucagonreceptor agonist. In another embodiment, OXM is a biologically activefragment of OXM. In another embodiment, biologically active OXM extendsfrom amino acid 30 to amino acid 37 of SEQ ID NO: 1. In anotherembodiment, biologically active OXM extends from amino acid 19 to aminoacid 37 of SEQ ID NO: 1. In another embodiment, OXM of the inventioncorresponds to an octapeptide from which the two C-terminal amino acidsare deleted. In another embodiment, OXM of the invention corresponds toany fragment of SEQ ID NO: 1 which retains OXM activity as providedherein.

In one embodiment, OXM refers to a peptide homologue of the peptide ofSEQ ID NO: 1. In one embodiment, OXM amino acid sequence of the presentinvention is at least 50% homologous to the OXM sequence set forth inSEQ ID NO: 1 as determined using BlastP software of the National Centerof Biotechnology Information (NCBI) using default parameters. In oneembodiment, OXM amino acid sequence of the present invention is at least60% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 70% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 80% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 90% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.In one embodiment, OXM amino acid sequence of the present invention isat least 95% homologous to the OXM sequence set forth in SEQ ID NO: 1 asdetermined using BlastP software of the NCBI using default parameters.

In one embodiment, the long-acting OXM of the invention maintains thebiological activity of unmodified OXM. In another embodiment, thelong-acting OXM of the invention comprising OXM biological activity. Inanother embodiment, the biological activity of a long-acting OXM of theinvention comprises reducing digestive secretions. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reducing and delaying gastric emptying. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises the inhibition of the fed motility pattern in thesmall intestine. In another embodiment, the biological activity of along-acting OXM of the invention comprises the inhibition of acidsecretion stimulated by pentagastrin. In another embodiment, thebiological activity of a long-acting OXM of the invention comprises anincrease of gastric somatostatin release. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisespotentiating the effects of peptide YY. In another embodiment, thebiological activity of a long-acting OXM of the invention comprises theinhibition of ghrelin release. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises the stimulationof aminopyrine accumulation and cAMP production. In another embodiment,the biological activity of a long-acting OXM of the invention comprisesbinding the GLP-1 receptor. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises binding theGlucagon receptor. In another embodiment, the biological activity of along-acting OXM of the invention comprises stimulating H+ production byactivating the adenylate cyclase. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises inhibitinghistamine-stimulated gastric acid secretion. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisesinhibiting food intake. In another embodiment, the biological activityof a long-acting OXM of the invention comprises stimulating insulinrelease. In another embodiment, the biological activity of a long-actingOXM of the invention comprises inhibiting exocrine pancreatic secretion.In another embodiment, the biological activity of a long-acting OXM ofthe invention comprises increasing insulin sensitivity. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reducing glucose levels.

In one embodiment, the present invention further provides a method forextending the biological half-life of oxyntomodulin, consisting of thestep of conjugating oxyntomodulin, a polyethylene glycol polymer (PEGpolymer) and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein, in another embodiment, the PEG polymer is conjugated to aLysine residue on position number 12 or to a Lysine residue on positionnumber 30 or to the amino terminus of the oxyntomodulin's amino acidsequence via 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 12 of the oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to a Lysine residue on positionnumber 30 of said oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another embodiment, the invention relates to a method for extendingthe biological half-life of oxyntomodulin, consisting of the step ofconjugating oxyntomodulin, a polyethylene glycol polymer (PEG polymer)and 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS) in a molar ratio of 1:1:1,wherein said PEG polymer is conjugated to the amino terminus of saidoxyntomodulin's amino acid sequence via 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the molar ratio of OXM-PEG- and linker is1:1:1-1:1:3.5. In another embodiment, the molar ratio is 1:1:1-1:1:10.0.In another embodiment, the higher ratio of linker allows for optimizedyield of the composition.

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 or to the Lysine residue on positionnumber 30 or to the amino terminus of the oxyntomodulin's amino acidsequence via 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the invention relates to a method of improvingthe area under the curve (AUC) of oxyntomodulin, consisting of the stepof conjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 12 of the oxyntomodulin's amino acid sequencevia 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the Lysineresidue on position number 30 of the oxyntomodulin's amino acid sequencevia 9-fluorenylmethoxycarbonyl (Fmoc) or2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In one embodiment, the invention relates to a method of improving thearea under the curve (AUC) of oxyntomodulin, consisting of the step ofconjugating a polyethylene glycol polymer (PEG polymer) to the aminoterminus of the oxyntomodulin's amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In one aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 or to the Lysine residue on position number 30 or tothe amino terminus of the oxyntomodulis amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 12 of the oxyntomodulis amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the Lysine residue onposition number 30 of the oxyntomodulis amino acid sequence via9-fluorenylmethoxycarbonyl (Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl(FMS).

In another aspect, provided herein is a method of reducing the dosingfrequency of oxyntomodulin, consisting of the step of conjugating apolyethylene glycol polymer (PEG polymer) to the amino terminus of theoxyntomodulis amino acid sequence via 9-fluorenylmethoxycarbonyl (Fmoc)or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS).

In another embodiment, the present invention further provides a methodfor reducing food intake, in a subject, comprising the step ofadministering to the subject a compositing consisting of oxyntomodulinconjugated to polyethylene glycol polymer (PEG polymer) via a flexiblelinker, wherein said flexible linker is 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), and wherein said PEGpolymer is conjugated to a Lysine residue on position number 12 or to aLysine residue on position number 30 or to the amino terminus of theoxyntomodulin's amino acid sequence via the Fmoc or the FMS.

In another embodiment, the present invention further provides a methodfor reducing body weight in a subject, comprising the step ofadministering to the subject a compositing consisting of oxyntomodulinconjugated to polyethylene glycol polymer (PEG polymer) via a flexiblelinker, wherein said flexible linker is 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), and wherein said PEGpolymer is conjugated to a Lysine residue on position number 12 or to aLysine residue on position number 30 or to the amino terminus of theoxyntomodulin's amino acid sequence via the Fmoc or the FMS.

In another embodiment, the present invention further provides a methodfor inducing glycemic control in a subject, comprising the step ofadministering to the subject a compositing consisting of oxyntomodulinconjugated to polyethylene glycol polymer (PEG polymer) via a flexiblelinker, wherein said flexible linker is 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), and wherein said PEGpolymer is conjugated to a Lysine residue on position number 12 or to aLysine residue on position number 30 or to the amino terminus of theoxyntomodulin's amino acid sequence via the Fmoc or the FMS.

The amino variant provided herein unexpectedly achieves reduced foodintake, weight control and glycemic control, as exemplified herein (seeExample 5). In one embodiment, the PEG modification of the OXM peptideprovided herein unexpectedly does not interfere with OXM function.

In another embodiment, the present invention provides a method forimproving cholesterol levels in a subject, comprising the step ofadministering to the subject an effective amount of a compositionprovided herein. In another embodiment, improving cholesterol levelscomprises reducing LDL cholesterol while increasing HDL cholesterol in asubject. In another embodiment, LDL cholesterol levels are reduced tobelow 200 mg/dL, but above 0 mg/dL. In another embodiment, LDLcholesterol levels are reduced to about 100-129 mg/dL. In anotherembodiment, LDL cholesterol levels are reduced to below 100 mg/dL, butabove 0 mg/dL. In another embodiment, LDL cholesterol levels are reducedto below 70 mg/dL, but above 0 mg/dL. In another embodiment, LDLcholesterol levels are reduced to below 5.2 mmol/L, but above 0 mmol/L.In another embodiment, LDL cholesterol levels are reduced to about 2.6to 3.3 mmol/L. In another embodiment, LDL cholesterol levels are reducedto below 2.6 mmol/L, but above 0 mmol/L. In another embodiment, LDLcholesterol levels are reduced to below 1.8 mmol/L, but above 0 mmol/L.

In another embodiment, the present invention further provides a methodfor reducing insulin resistance in a subject, comprising the step ofadministering to the subject an effective amount of a compositionprovided herein.

In another embodiment, the biological activity of a long-acting OXM ofthe invention comprises inhibiting pancreatic secretion through a vagalneural indirect mechanism. In another embodiment, the biologicalactivity of a long-acting OXM of the invention comprises reducinghydromineral transport through the small intestine. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises stimulating glucose uptake. In another embodiment,the biological activity of a long-acting OXM of the invention comprisescontrolling/stimulating somatostatin secretion. In another embodiment,the biological activity of a long-acting OXM of the invention comprisesreduction in both food intake and body weight gain. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises reduction in adiposity. In another embodiment, thebiological activity of a long-acting OXM of the invention comprisesappetite suppression. In another embodiment, the biological activity ofa long-acting OXM of the invention comprises induction of anorexia. Inanother embodiment, the biological activity of a long-acting OXM of theinvention comprises reducing body weight in overweight and obesesubjects. In another embodiment, the biological activity of along-acting OXM of the invention comprises inducing changes in thelevels of the adipose hormones leptin and adiponectin. In anotherembodiment, the biological activity of a long-acting OXM of theinvention comprises increasing energy expenditure in addition todecreasing energy intake in overweight and obese subjects.

In another embodiment, a PEG polymer is attached to the amino terminusor lysine residue of oxyntomodulin via Fmoc or FMS. In anotherembodiment, the terms “attached” and “linked” are use interchangeably.In another embodiment, the PEG polymer is linked to the α-amino sidechain of OXM. In another embodiment, the PEG polymer is linked to theε-amino side chain of OXM. In another embodiment, the PEG polymer islinked to one or more ε-amino side chain of OXM. In another embodiment,the PEG polymer comprises a sulfhydryl moiety.

In another embodiment, PEG is linear. In another embodiment, PEG isbranched. In another embodiment, PEG has a molecular weight in the rangeof 200 to 200,000 Da. In another embodiment, PEG has a molecular weightin the range of 5,000 to 80,000 Da. In another embodiment, PEG has amolecular weight in the range of 5,000 to 40,000 Da. In anotherembodiment, PEG has a molecular weight in the range of 20,000 Da to40,000 Da.

In another embodiment, a long-acting OXM is prepared using PEGylatingagents, meaning any PEG derivative which is capable of reacting with afunctional group such as, but not limited to, NH₂, OH, SH, COOH, CHO,—N═C═O, —N═C═S, —SO₂Cl, —SO₂CH═CH₂, —PO₂Cl, —(CH₂)xHal, present at thefluorene ring of the Fmoc or FMS moiety. In another embodiment, thePEGylating agent is usually used in its mono-methoxylated form whereonly one hydroxyl group at one terminus of the PEG molecule is availablefor conjugation. In another embodiment, a bifunctional form of PEG whereboth termini are available for conjugation may be used if, for example,it is desired to obtain a conjugate with two peptide or protein residuescovalently attached to a single PEG moiety.

In another embodiment, branched PEGs are represented as R(PEG-OH)_(m) inwhich R represents a central core moiety such as pentaerythritol orglycerol, and m represents the number of branching arms. The number ofbranching arms (m) can range from three to a hundred or more. In anotherembodiment, the hydroxyl groups are subject to chemical modification. Inanother embodiment, branched PEG molecules are described in U.S. Pat.Nos. 6,113,906, 5,919,455, 5,643,575, and 5,681,567, which are herebyincorporated by reference in their entirety.

In another embodiment, the present invention provides OXM with a PEGmoiety which is not attached directly to the OXM, as in the standardpegylation procedure, but rather the PEG moiety is attached through alinker such as Fmoc or FMS. In another embodiment, the linker is highlysensitive to bases and is removable under mild basic conditions. Inanother embodiment, OXM connected to PEG via Fmoc or FMS is equivalentlyactive to the free OXM. In another embodiment, OXM connected to PEG viaFmoc or FMS is more active than the free OXM. In another embodiment, OXMconnected to PEG via Fmoc or FMS comprises different activity than thefree OXM. In another embodiment, OXM connected to PEG via Fmoc or FMSunlike the free OXM, has no central nervous system activity. In anotherembodiment, OXM connected to PEG via Fmoc or FMS unlike the free OXM,can not enter the brain through the blood brain barrier. In anotherembodiment, OXM connected to PEG via Fmoc or FMS comprises extendedcirculation half-life compared to the free OXM. In another embodiment,OXM connected to PEG via Fmoc or FMS loses its PEG moiety together withthe Fmoc or FMS moiety thus recovering the free OXM.

In another embodiment, the present invention provides a compound of theformula: (X)n-Y, wherein Y is a moiety of OXM bearing a free amino,carboxyl, or hydroxyl and X is a radical of formula (i):

In another embodiment, R₁ is a radical containing a protein or polymercarrier moiety; polyethylene glycol (PEG) moiety; R₂ is selected fromthe group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl,alkaryl, aralkyl, halogen, nitro, —SO₃H, —SO₂NHR, amino, ammonium,carboxyl, PO₃H.sub.2, and OPO₃H₂; R is selected from the groupconsisting of hydrogen, alkyl and aryl; R₃ and R₄, the same ordifferent, are each selected from the group consisting of hydrogen,alkyl and aryl; A is a covalent bond when the radical is linked to anamino or hydroxyl group of the OXM-Y; n is an integer of at least one,and pharmaceutically acceptable salts thereof.

In another embodiment, the terms “alkyl”, “alkoxy”, “alkoxyalkyl”,“aryl”, “alkaryl” and “aralkyl” are used to denote alkyl radicals of1-8, preferably 1-4 carbon atoms, e.g. methyl, ethyl, propyl, isopropyland butyl, and aryl radicals of 6-10 carbon atoms, e.g. phenyl andnaphthyl. The term “halogen” includes bromo, fluoro, chloro and iodo.

In another embodiment, R₂, R₃ and R₄ are each hydrogen and A is —OCO—,namely the 9-fluorenylmethoxycarbonyl radical (hereinafter “Fmoc”). Inanother embodiment, R is —SO₃H at position 2 of the fluorene ring, R₃and R₄ are each hydrogen, and A is —OCO—, namely the2-sulfo-9-fluorenylmethoxycarbonyl radical (hereinafter “FMS”).

In another embodiment, pegylation of OXM and preparation of the(PEG-Fmoc)n-OXM or (PEG-FMS)n-OXM conjugates includes attachingMAL-FMS-NHS or MAL-Fmoc-NHS to the amine component of OXM, thusobtaining a MAL-FMS-OXM or MAL-Fmoc-OXM conjugate, and then substitutingPEG-SH for the maleimide moiety, producing the (PEG-FMS)n-OXM or(PEG-Fmoc)n-OXM conjugate, respectively.

In another embodiment, pegylation of OXM includes reacting MAL-FMS-NHSor MAL-Fmoc-NHS with PEG-SH, thus forming a PEG-FMS-NHS or PEG-Fmoc-NHSconjugate, and then reacting it with the amine component of OXMresulting in the desired (PEG-FMS)n-OXM or (PEG-Fmoc)n-OXM conjugate,respectively. In another embodiment, pegylation of peptides/proteinssuch as OXM are described in U.S. Pat. No. 7,585,837, which isincorporated herein by reference in its entirety. In another embodiment,reverse-pegylation of peptides/proteins such as OXM with Fmoc or FMS aredescribed in U.S. Pat. No. 7,585,837.

In another embodiment, the phrases “long acting OXM” and “reversepegylated OXM” are used interchangeably. In another embodiment, reversepegylated OXM is composed of PEG-FMS-OXM and PEG-Fmoc-OXM hereinidentified by the formulas: (PEG-FMS)n-OXM or (PEG-Fmoc)n-OXM, wherein nis an integer of at least one, and OXM is linked to the FMS or Fmocradical through at least one amino group.

In another embodiment, the conjugation of PEG-Fmoc or PEG-FMS to Lys12or Lys30 or the amino terminus of OXM does not render the OXM inactive.

In one embodiment, the Lys12 variant is more effective at providingweight control than the other variants provided herein. In anotherembodiment, the Lys30 variant provided herein is more effective atachieving weight control than the other variants provided herein. Inanother embodiment, the amino variant provided herein is more effectiveat achieving weight control than the other variants provided herein.

In one embodiment, the Lys12 variant is more effective at achievingchronic glycemic control than the other variants provided herein. Inanother embodiment, the Lys30 variant provided herein is more effectiveat achieving chronic glycemic control than the other variants providedherein. In another embodiment, the amino variant provided herein is moreeffective at achieving glycemic control than the other variants providedherein.

Therapeutic Uses

In another embodiment, PEG-Fmoc-OXM and PEG-FMS-OXM and pharmaceuticalcompositions comprising them are utilized for the prevention ofhyperglycemia, for improving glycemic control, for treatment of diabetesmellitus selected from the group consisting of non-insulin dependentdiabetes mellitus (in one embodiment, Type 2 diabetes),insulin-dependent diabetes mellitus (in one embodiment, Type 1diabetes), and gestational diabetes mellitus, or any combinationthereof. In another embodiment, PEG-Fmoc-OXM and PEG-FMS-OXM andpharmaceutical compositions comprising them are utilized for treatingType 2 Diabetes. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themare utilized for increasing sensitivity to insulin. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them are utilized forreducing insulin resistance.

In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them areutilized for the suppression of appetite. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them are utilized for inducing satiety. Inanother embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them are utilized forthe reduction of body weight. In another embodiment, the PEG-Fmoc-OXMand PEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for the reduction of body fat. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them are utilized for thereduction of body mass index. In another embodiment, the PEG-Fmoc-OXMand PEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for the reduction of food consumption. Inanother embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them are utilized fortreating obesity. In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for treating diabetes mellitus associatedwith obesity. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themare utilized for increasing heart rate. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them are utilized for increasing the basalmetabolic rate (BMR). In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for increasing energy expenditure. Inanother embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them are utilized forinducing glucose tolerance. In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for inducing glycemic control. In oneembodiment, glycemic control refers to non-high and/or non-fluctuatingblood glucose levels and/or non-high and/or non-fluctuating glycosylatedhemoglobin levels.

In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them areutilized for inhibiting weight increase, where in another embodiment,the weight increase is due to fat increase. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them are utilized for reducing blood glucoselevels. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them areutilized for decreasing caloric intake. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them are utilized for decreasing appetite. Inanother embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them are utilized forweight control. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themare utilized for inducing or promoting weight loss. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them are utilized formaintaining any one or more of a desired body weight, a desired BodyMass Index, a desired appearance and good health. In another embodiment,PEG the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein andpharmaceutical compositions comprising them are utilized for controllinga lipid profile. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themare utilized for reducing triglyceride levels. In another embodiment,the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein andpharmaceutical compositions comprising them are utilized for reducingglycerol levels. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themare utilized for increasing adiponectin levels. In another embodiment,the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein andpharmaceutical compositions comprising them are utilized for reducingfree fatty acid levels.

In one embodiment, the terms “reducing the level of” refers to areduction of about 1-10% relative to an original, wild-type, normal orcontrol level. In another embodiment, the reduction is of about 11-20%.In another embodiment, the reduction is of about 21-30%. In anotherembodiment, the reduction is of about 31-40%. In another embodiment, thereduction is of about 41-50%. In another embodiment, the reduction is ofabout 51-60%. In another embodiment, the reduction is of about 61-70%.In another embodiment, the reduction is of about 71-80%. In anotherembodiment, the reduction is of about 81-90%. In another embodiment, thereduction is of about 91-95%. In another embodiment, the reduction is ofabout 96-100%.

In one embodiment, the terms “increasing the level of” or “extending”refers to a increase of about 1-10% relative to an original, wild-type,normal or control level. In another embodiment, the increase is of about11-20%. In another embodiment, the increase is of about 21-30%. Inanother embodiment, the increase is of about 31-40%. In anotherembodiment, the increase is of about 41-50%. In another embodiment, theincrease is of about 51-60%. In another embodiment, the increase is ofabout 61-70%. In another embodiment, the increase is of about 71-80%. Inanother embodiment, the increase is of about 81-90%. In anotherembodiment, the increase is of about 91-95%. In another embodiment, theincrease is of about 96-100%.

In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them areutilized for reducing cholesterol levels. In one embodiment, thereduction in cholesterol levels is greater than the reduction observedafter administration of native OXM. In one embodiment, the PEG-Fmoc-OXMand PEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them lower cholesterol levels by 60-70%. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them lower cholesterol levelsby 50-100%. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXMvariants provided herein and pharmaceutical compositions comprising themlower cholesterol levels by 25-90%. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them lower cholesterol levels by 50-80%. Inanother embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them lower cholesterollevels by 40-90%. In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them are utilized for increasing HDL cholesterol levels.

In one embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them may be used forthe purposes described herein without a significant decrease ineffectiveness over the course of administration. In one embodiment,PEG-Fmoc-OXM and PEG-FMS-OXM and pharmaceutical compositions comprisingthem remains effective for 1 day. In another embodiment, PEG-Fmoc-OXMand PEG-FMS-OXM and pharmaceutical compositions comprising them remainseffective for 2-6 days. In one embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them remains effective for 1 week. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them remain effective for 2 weeks. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them remain effective for 3weeks. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them remaineffective for 4 weeks. In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them remain effective for 6 weeks. In another embodiment, thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them remain effective for 2 months. In anotherembodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants provided hereinand pharmaceutical compositions comprising them remain effective for 4months. In another embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variantsprovided herein and pharmaceutical compositions comprising them remaineffective for 6 months. In another embodiment, the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them remain effective for 1 year or more.

In one embodiment, the PEG-Fmoc-OXM and PEG-FMS-OXM variants providedherein and pharmaceutical compositions comprising them may be used forthe purposes described herein and may be effective immediately uponadministration of the first dose. In another embodiment, PEG thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them are effective after two or more doses havebeen administered.

In another embodiment, methods of utilizing the PEG-Fmoc-OXM andPEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them as described hereinabove are applied to a human subjectafflicted with a disease or condition that can be alleviated, inhibited,and/or treated by OXM. In another embodiment, methods of utilizing thePEG-Fmoc-OXM and PEG-FMS-OXM variants provided herein and pharmaceuticalcompositions comprising them as described hereinabove are veterinarymethods. In another embodiment, methods of utilizing the PEG-Fmoc-OXMand PEG-FMS-OXM variants provided herein and pharmaceutical compositionscomprising them as described hereinabove are applied to animals such asfarm animals, pets, and lab animals. Thus, in one embodiment, a subjectof the present invention is feline, canine, bovine, porcine, murine,aquine, etc.

In another embodiment, the present invention provides a method oftreating or reducing a disease treatable or reducible by OXM or apharmaceutical formulation comprising the same, in a subject, comprisingthe step of administering to a subject a therapeutically effectiveamount of the PEG-Fmoc-OXM and/or PEG-FMS-OXM variants provided herein,thereby treating or reducing a disease treatable or reducible by OXM ina subject.

In another embodiment, OXM, “peptide” or “protein” as used hereinencompasses native peptides (either degradation products, syntheticallysynthesized proteins or recombinant proteins) and peptidomimetics(typically, synthetically synthesized proteins), as well as peptoids andsemipeptoids which are protein analogs, which have, in some embodiments,modifications rendering the proteins even more stable while in a body ormore capable of penetrating into cells.

In another embodiment, a “PEG-Fmoc-OXM and/or a PEG-FMS-OXM variant” isa PEG-Fmoc-OXM and/or a PEG-FMS-OXM variant whereby either FMS or Fmocis bound to OXM at Lys12. In another embodiment, the PEG-Fmoc-OXM and/ora PEG-FMS-OXM variant is a PEG-Fmoc-OXM and/or a PEG-FMS-OXM variantwherein either FMS or Fmoc is bound to OXM at Lys30. In anotherembodiment, the PEG-Fmoc-OXM and/or a PEG-FMS-OXM variant is aPEG-Fmoc-OXM and/or a PEG-FMS-OXM variant whereby either FMS or Fmoc isbound to OXM at the amino terminus.

In another embodiment, modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

In another embodiment, peptide bonds (—CO—NH—) within the peptide aresubstituted. In some embodiments, the peptide bonds are substituted byN-methylated bonds (—N(CH3)-CO—). In another embodiments, the peptidebonds are substituted by ester bonds (—C(R)H—C—O—O—C(R)—N—). In anotherembodiment, the peptide bonds are substituted by ketomethylen bonds(—CO—CH2-). In another embodiment, the peptide bonds are substituted byα-aza bonds (—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carbabonds (—CH2-NH—). In another embodiments, the peptide bonds aresubstituted by hydroxyethylene bonds (—CH(OH)—CH2-). In anotherembodiment, the peptide bonds are substituted by thioamide bonds(—CS—NH—). In some embodiments, the peptide bonds are substituted byolefinic double bonds (—CH═CH—). In another embodiment, the peptidebonds are substituted by retro amide bonds (—NH—CO—). In anotherembodiment, the peptide bonds are substituted by peptide derivatives(—N(R)—CH2-CO—), wherein R is the “normal” side chain, naturallypresented on the carbon atom. In some embodiments, these modificationsoccur at any of the bonds along the peptide chain and even at several(2-3 bonds) at the same time.

In one embodiment, natural aromatic amino acids of the protein such asTrp, Tyr and Phe, are substituted for synthetic non-natural acid such asPhenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivativesof Phe, halogenated derivatives of Phe or o-methyl-Tyr. In anotherembodiment, the peptides of the present invention include one or moremodified amino acid or one or more non-amino acid monomers (e.g. fattyacid, complex carbohydrates etc).

In comparison to the wild-type OXM, the OXM derivatives or variants ofthe present invention contain several amino acid substitutions, and/orcan be PEGylated or otherwise modified (e.g. recombinantly orchemically).

The OXM provided herein also covers any analogue of the above OXMsequence. Any one or more amino acid residues in the sequence can beindependently replaced with a conservative replacement as well known inthe art i.e. replacing an amino acid with one of a similar chemical typesuch as replacing one hydrophobic amino acid with another.Alternatively, non-conservative amino acid mutations can be made thatresult in an enhanced effect or biological activity of OXM. In oneembodiment, the OXM is modified to be resistant to cleavage andinactivation by dipeptidyl peptidase IV (DPP-IV). Derivatives, andvariants of OXM and methods of generating the same are disclosed in USPatent Application Publication Nos. 2011/0152182, US Patent ApplicationPublication Nos. 2011/0034374, US Patent Application Publication Nos.2010/0144617, all of which are incorporated by reference herein.

In one embodiment, the dual GLP-1/Glucagon receptor agonist providedherein can be chemically modified. In another embodiment, the OXMprovided herein can be chemically modified. In particular, the aminoacid side chains, the amino terminus and/or the carboxy acid terminus ofOXM can be modified. For example, the OXM can undergo one or more ofalkylation, disulphide formation, metal complexation, acylation,esterification, amidation, nitration, treatment with acid, treatmentwith base, oxidation or reduction. Methods for carrying out theseprocesses are well known in the art. In particular the OXM is providedas a lower alkyl ester, a lower alkyl amide, a lower dialkyl amide, anacid addition salt, a carboxylate salt or an alkali addition saltthereof. In particular, the amino or carboxylic termini of the OXM maybe derivatised by for example, esterification, amidation, acylation,oxidation or reduction. In particular, the carboxylic terminus of theOXM can be derivatised to form an amide moiety.

In one embodiment, “amino acid” or “amino acids” is understood toinclude the 20 naturally occurring amino acids; those amino acids oftenmodified post-translationally in vivo, including, for example,hydroxyproline, phosphoserine and phosphothreonine; and other unusualamino acid including, but not limited to, 2-aminoadipic acid,hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. Inone embodiment, “amino acid” includes both D- and L-amino acids. It isto be understood that other synthetic or modified amino acids can bealso be used.

In one embodiment, the OXM of the present invention are utilized intherapeutics which requires OXM to be in a soluble form. In anotherembodiment, OXM of the present invention includes one or morenon-natural or natural polar amino acid, including, but not limited to,serine and threonine which are capable of increasing protein solubilitydue to their hydroxyl-containing side chain.

In one embodiment, OXM of present invention is biochemically synthesizedsuch as by using standard solid phase techniques. In another embodiment,these biochemical methods include exclusive solid phase synthesis,partial solid phase synthesis, fragment condensation, or classicalsolution synthesis.

In one embodiment, solid phase OXM synthesis procedures are well knownto one skilled in the art and further described by John Morrow Stewartand Janis Dillaha Young, Solid Phase Protein Syntheses (2nd Ed., PierceChemical Company, 1984). In another embodiment, synthetic proteins arepurified by preparative high performance liquid chromatography[Creighton T. (1983) Proteins, structures and molecular principles. WHFreeman and Co. N.Y.] and the composition of which can be confirmed viaamino acid sequencing by methods known to one skilled in the art.

In another embodiment, recombinant protein techniques are used togenerate the OXM of the present invention. In some embodiments,recombinant protein techniques are used for the generation of largeamounts of the OXM of the present invention. In another embodiment,recombinant techniques are described by Bitter et al., (1987) Methods inEnzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al.(1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J. 3:1671-1680 andBrogli et al., (1984) Science 224:838-843, Gurley et al. (1986) Mol.Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp 421-463.

In another embodiment, OXM of the present invention is synthesized usinga polynucleotide encoding OXM of the present invention. In someembodiments, the polynucleotide encoding OXM of the present invention isligated into an expression vector, comprising a transcriptional controlof a cis-regulatory sequence (e.g., promoter sequence). In someembodiments, the cis-regulatory sequence is suitable for directingconstitutive expression of the OXM of the present invention.

In one embodiment, the phrase “a polynucleotide” refers to a single ordouble stranded nucleic acid sequence which be isolated and provided inthe form of an RNA sequence, a complementary polynucleotide sequence(cDNA), a genomic polynucleotide sequence and/or a compositepolynucleotide sequences (e.g., a combination of the above).

In one embodiment, “complementary polynucleotide sequence” refers to asequence, which results from reverse transcription of messenger RNAusing a reverse transcriptase or any other RNA dependent DNA polymerase.In one embodiment, the sequence can be subsequently amplified in vivo orin vitro using a DNA polymerase.

In one embodiment, “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus it represents acontiguous portion of a chromosome.

In one embodiment, “composite polynucleotide sequence” refers to asequence, which is at least partially complementary and at leastpartially genomic. In one embodiment, a composite sequence can includesome exonal sequences required to encode the peptide of the presentinvention, as well as some intronic sequences interposing there between.In one embodiment, the intronic sequences can be of any source,including of other genes, and typically will include conserved splicingsignal sequences. In one embodiment, intronic sequences include cisacting expression regulatory elements.

In one embodiment, polynucleotides of the present invention are preparedusing PCR techniques, or any other method or procedure known to oneskilled in the art. In some embodiments, the procedure involves theligation of two different DNA sequences (See, for example, “CurrentProtocols in Molecular Biology”, eds. Ausubel et al., John Wiley & Sons,1992).

In one embodiment, a variety of prokaryotic or eukaryotic cells can beused as host-expression systems to express the OXM of the presentinvention. In another embodiment, these include, but are not limited to,microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the protein coding sequence; yeast transformed withrecombinant yeast expression vectors containing the protein codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors, such asTi plasmid, containing the protein coding sequence.

In one embodiment, non-bacterial expression systems are used (e.g.mammalian expression systems such as CHO cells) to express the OXM ofthe present invention. In one embodiment, the expression vector used toexpress polynucleotides of the present invention in mammalian cells ispCI-DHFR vector comprising a CMV promoter and a neomycin resistancegene.

In another embodiment, in bacterial systems of the present invention, anumber of expression vectors can be advantageously selected dependingupon the use intended for the protein expressed. In one embodiment,large quantities of OXM are desired. In one embodiment, vectors thatdirect the expression of high levels of the protein product, possibly asa fusion with a hydrophobic signal sequence, which directs the expressedproduct into the periplasm of the bacteria or the culture medium wherethe protein product is readily purified are desired. In one embodiment,certain fusion protein engineered with a specific cleavage site to aidin recovery of the protein. In one embodiment, vectors adaptable to suchmanipulation include, but are not limited to, the pET series of E. coliexpression vectors [Studier et al., Methods in Enzymol. 185:60-89(1990)].

In one embodiment, yeast expression systems are used. In one embodiment,a number of vectors containing constitutive or inducible promoters canbe used in yeast as disclosed in U.S. Pat. No. 5,932,447. In anotherembodiment, vectors which promote integration of foreign DNA sequencesinto the yeast chromosome are used.

In one embodiment, the expression vector of the present invention canfurther include additional polynucleotide sequences that allow, forexample, the translation of several proteins from a single mRNA such asan internal ribosome entry site (IRES) and sequences for genomicintegration of the promoter-chimeric protein.

In one embodiment, mammalian expression vectors include, but are notlimited to, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB,pNMT1, pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

In another embodiment, expression vectors containing regulatory elementsfrom eukaryotic viruses such as retroviruses are used by the presentinvention. SV40 vectors include pSVT7 and pMT2. In another embodiment,vectors derived from bovine papilloma virus include pBV-1MTHA, andvectors derived from Epstein Bar virus include pHEBO, and p2O5. Otherexemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5,baculovirus pDSVE, and any other vector allowing expression of proteinsunder the direction of the SV-40 early promoter, SV-40 later promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

In one embodiment, plant expression vectors are used. In one embodiment,the expression of OXM coding sequence is driven by a number ofpromoters. In another embodiment, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514(1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J.6:307-311 (1987)] are used. In another embodiment, plant promoters areused such as, for example, the small subunit of RUBISCO [Coruzzi et al.,EMBO J. 3:1671-1680 (1984); and Brogli et al., Science 224:838-843(1984)] or heat shock promoters, e.g., soybean hsp17.5-E or hsp17.3-B[Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment,constructs are introduced into plant cells using Ti plasmid, Ri plasmid,plant viral vectors, direct DNA transformation, microinjection,electroporation and other techniques well known to the skilled artisan.See, for example, Weissbach & Weissbach [Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Otherexpression systems such as insects and mammalian host cell systems,which are well known in the art, can also be used by the presentinvention.

It will be appreciated that other than containing the necessary elementsfor the transcription and translation of the inserted coding sequence(encoding the protein), the expression construct of the presentinvention can also include sequences engineered to optimize stability,production, purification, yield or activity of the expressed protein.

Various methods, in some embodiments, can be used to introduce theexpression vector of the present invention into the host cell system. Insome embodiments, such methods are generally described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (1989, 1992), in Ausubel et al., Current Protocolsin Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Changet al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vegaet al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Butterworths, BostonMass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

In one embodiment, transformed cells are cultured under effectiveconditions, which allow for the expression of high amounts ofrecombinant OXM. In another embodiment, effective culture conditionsinclude, but are not limited to, effective media, bioreactor,temperature, pH and oxygen conditions that permit protein production. Inone embodiment, an effective medium refers to any medium in which a cellis cultured to produce the recombinant OXM of the present invention. Inanother embodiment, a medium typically includes an aqueous solutionhaving assimilable carbon, nitrogen and phosphate sources, andappropriate salts, minerals, metals and other nutrients, such asvitamins. In one embodiment, cells of the present invention can becultured in conventional fermentation bioreactors, shake flasks, testtubes, microtiter dishes and petri plates. In another embodiment,culturing is carried out at a temperature, pH and oxygen contentappropriate for a recombinant cell. In another embodiment, culturingconditions are within the expertise of one of ordinary skill in the art.

In one embodiment, depending on the vector and host system used forproduction, resultant OXM of the present invention either remain withinthe recombinant cell, secreted into the fermentation medium, secretedinto a space between two cellular membranes, such as the periplasmicspace in E. coli; or retained on the outer surface of a cell or viralmembrane.

In one embodiment, following a predetermined time in culture, recoveryof the recombinant OXM is effected.

In one embodiment, the phrase “recovering the recombinant OXM” usedherein refers to collecting the whole fermentation medium containing theOXM and need not imply additional steps of separation or purification.

In one embodiment, OXM of the present invention is purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

In one embodiment, to facilitate recovery, the expressed coding sequencecan be engineered to encode the protein of the present invention andfused cleavable moiety. In one embodiment, a fusion protein can bedesigned so that the protein can be readily isolated by affinitychromatography; e.g., by immobilization on a column specific for thecleavable moiety. In one embodiment, a cleavage site is engineeredbetween the protein and the cleavable moiety and the protein can bereleased from the chromatographic column by treatment with anappropriate enzyme or agent that specifically cleaves the fusion proteinat this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988);and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)]. In anotherembodiment, the OXM of the present invention is retrieved in“substantially pure” form. In another embodiment, the phrase“substantially pure” refers to a purity that allows for the effectiveuse of the OXM in the applications described herein.

In one embodiment, the OXM of the present invention can also besynthesized using in vitro expression systems. In one embodiment, invitro synthesis methods are well known in the art and the components ofthe system are commercially available.

In another embodiment, in vitro binding activity is ascertained bymeasuring the ability of native, recombinant and/or reverse pegylatedOXM as described herein as well as pharmaceutical compositionscomprising the same to treat or ameliorate diseases or conditions suchas but not limited to: diabetes mellitus, obesity, eating disorders,metabolic disorders, etc. In another embodiment, in vivo activity isdeduced by known measures of the disease that is being treated.

In another embodiment, a dose of OXM peptide of the present inventioncomprises from 0.005 to 0.1 milligrams/kg in an injectable solution. Inanother embodiment, the comprises from 0.005 to 0.5 milligrams/kg OXMpeptide. In another embodiment, the dose comprises from 0.05 to 0.1micrograms OXM peptide. In another embodiment, the dose comprises from0.005 to 0.1 milligrams/kg OXM peptide in an injectable solution.

In another embodiment, a dose of reverse pegylated OXM is administeredonce a day. In another embodiment, a dose of reverse pegylated OXM isadministered once every 36 hours. In another embodiment, a dose ofreverse pegylated OXM is administered once every 48 hours. In anotherembodiment, a dose of reverse pegylated OXM is administered once every60 hours. In another embodiment, a dose of reverse pegylated OXM isadministered once every 72 hours. In another embodiment, a dose ofreverse pegylated OXM is administered once every 84 hours. In anotherembodiment, a dose of reverse pegylated OXM is administered once every96 hours. In another embodiment, a dose of reverse pegylated OXM isadministered once every 5 days. In another embodiment, a dose of reversepegylated OXM is administered once every 6 days. In another embodiment,a dose of reverse pegylated OXM is administered once every 7 days. Inanother embodiment, a dose of reverse pegylated OXM is administered onceevery 8-10 days. In another embodiment, a dose of reverse pegylated OXMis administered once every 10-12 days. In another embodiment, a dose ofreverse pegylated OXM is administered once every 12-15 days. In anotherembodiment, a dose of reverse pegylated OXM is administered once every15-25 days.

In another embodiment, reverse pegylated OXM of the present invention isadministered by an intramuscular (IM) injection, subcutaneous (SC)injection, or intravenous (IV) injection once a week.

In another embodiment, the reverse pegylated OXM of the presentinvention can be provided to the individual per se. In one embodiment,the reverse pegylated OXM of the present invention can be provided tothe individual as part of a pharmaceutical composition where it is mixedwith a pharmaceutically acceptable carrier.

In another embodiment, a “pharmaceutical composition” refers to apreparation of long-acting OXN as described herein with other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of a pharmaceutical composition is to facilitate administrationof a compound to an organism. In another embodiment, a reverse pegylatedOXM is accountable for the biological effect.

In another embodiment, any of the compositions of this invention willcomprise at least a reverse pegylated OXM. In one embodiment, thepresent invention provides combined preparations. In one embodiment, “acombined preparation” defines especially a “kit of parts” in the sensethat the combination partners as defined above can be dosedindependently or by use of different fixed combinations withdistinguished amounts of the combination partners i.e., simultaneously,concurrently, separately or sequentially. In some embodiments, the partsof the kit of parts can then, e.g., be administered simultaneously orchronologically staggered, that is at different time points and withequal or different time intervals for any part of the kit of parts. Theratio of the total amounts of the combination partners, in someembodiments, can be administered in the combined preparation. In oneembodiment, the combined preparation can be varied, e.g., in order tocope with the needs of a patient subpopulation to be treated or theneeds of the single patient which different needs can be due to aparticular disease, severity of a disease, age, sex, or body weight ascan be readily made by a person skilled in the art.

In another embodiment, the phrases “physiologically acceptable carrier”and “pharmaceutically acceptable carrier” which be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases. In one embodiment, one of the ingredients includedin the pharmaceutically acceptable carrier can be for examplepolyethylene glycol (PEG), a biocompatible polymer with a wide range ofsolubility in both organic and aqueous media (Mutter et al. (1979).

In another embodiment, “excipient” refers to an inert substance added toa pharmaceutical composition to further facilitate administration of along-acting OXN. In one embodiment, excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs are found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

In another embodiment, suitable routes of administration of the peptideof the present invention, for example, include oral, rectal,transmucosal, transnasal, intestinal or parenteral delivery, includingintramuscular, subcutaneous and intramedullary injections as well asintrathecal, direct intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections.

The present invention also includes reverse pegylated OXM for use in themanufacture of a medicament for administration by a route peripheral tothe brain for any of the methods of treatment described above. Examplesof peripheral routes include oral, rectal, parenteral e.g. intravenous,intramuscular, or intraperitoneal, mucosal e.g. buccal, sublingual,nasal, subcutaneous or transdermal administration, includingadministration by inhalation. Preferred dose amounts of OXM for themedicaments are given below.

The present invention provides a pharmaceutical composition comprisingreverse pegylated OXM and a pharmaceutically suitable carrier, in a formsuitable for oral, rectal, parenteral, e.g. intravenous, intramuscular,or intraperitoneal, mucosal e.g. buccal, sublingual, nasal, subcutaneousor transdermal administration, including administration by inhalation.If in unit dosage form, the dose per unit may be, for example, asdescribed below or as calculated on the basis of the per kg doses givenbelow.

In another embodiment, the preparation is administered in a local ratherthan systemic manner, for example, via injection of the preparationdirectly into a specific region of a patient's body. In anotherembodiment, a reverse pegylated OXM is formulated in an intranasaldosage form. In another embodiment, a reverse pegylated OXM isformulated in an injectable dosage form.

Various embodiments of dosage ranges are contemplated by this invention:the OXM peptide component within of the reverse pegylated OXMcomposition is administered in a range of 0.01-0.5 milligrams/kg bodyweight per 3 days (only the weight of the OXM within the reversepegylated OXM composition is provided as the size of PEG can differsubstantially). In another embodiment, the OXM peptide component withinof the reverse pegylated OXM composition is administered in a range of0.01-0.5 milligrams/kg body weight per 7 days. In another embodiment,the OXM peptide component within of the reverse pegylated OXMcomposition is administered in a range of 0.01-0.5 milligrams/kg bodyweight per 10 days. In another embodiment, the OXM peptide componentwithin of the reverse pegylated OXM composition is administered in arange of 0.01-0.5 milligrams/kg body weight per 14 days. In anotherembodiment, unexpectedly, the effective amount of OXM in a reversepegylated OXM composition is ¼- 1/10 of the effective amount of freeOXM. In another embodiment, unexpectedly, reverse pegylation of OXMenables limiting the amount of OXM prescribed to a patient by at least50% compared with free OXM. In another embodiment, unexpectedly, reversepegylation of OXM enables limiting the amount of OXM prescribed to apatient by at least 70% compared with free OXM. In another embodiment,unexpectedly, reverse pegylation of OXM enables limiting the amount ofOXM prescribed to a patient by at least 75% compared with free OXM. Inanother embodiment, unexpectedly, reverse pegylation of OXM enableslimiting the amount of OXM prescribed to a patient by at least 80%compared with free OXM. In another embodiment, unexpectedly, reversepegylation of OXM enables limiting the amount of OXM prescribed to apatient by at least 85% compared with free OXM. In another embodiment,unexpectedly, reverse pegylation of OXM enables limiting the amount ofOXM prescribed to a patient by at least 90% compared with free OXM.

In another embodiment, the OXM peptide component within of the reversepegylated OXM composition is administered in a range of 0.01-0.5milligrams/kg body weight once every 3 days (only the weight of the OXMwithin the reverse pegylated OXM composition is provided as the size ofPEG can differ substantially). In another embodiment, the OXM peptidecomponent within of the reverse pegylated OXM composition isadministered in a range of 0.01-0.5 milligrams/kg body weight once every7 days. In another embodiment, the OXM peptide component within of thereverse pegylated OXM composition is administered in a range of 0.01-0.5milligrams/kg body weight once every 10 days. In another embodiment, theOXM peptide component within of the reverse pegylated OXM composition isadministered in a range of 0.01-0.5 milligrams/kg body weight once every14 days.

In another embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 2-fold and reducesthe effective weekly dose by at least 2-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 3-fold and reducesthe effective weekly dose by at least 3-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 4-fold and reducesthe effective weekly dose by at least 4-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 5-fold and reducesthe effective weekly dose by at least 5-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, reverse pegylated OXM compared to free OXM bothreduces the effective dosing frequency by at least 6-fold and reducesthe effective weekly dose by at least 6-fold, thus limiting the risk ofadverse events and increasing compliance with the use of OXM therapy. Inanother embodiment, effective dosing frequency and effective weekly doseare based on: (1) the weight of administered OXM component within thereverse pegylated OXM composition; and (2) the weight of administeredOXM component within the free OXM (unmodified OXM) composition.

In another embodiment, the methods of the invention include increasingthe compliance of patients afflicted with chronic illnesses that are inneed of OXM therapy. In another embodiment, the methods of the inventionenable reduction in the dosing frequency of OXM by reverse pegylatingOXM as described hereinabove. In another embodiment, the methods of theinvention include increasing the compliance of patients in need of OXMtherapy by reducing the frequency of administration of OXM. In anotherembodiment, reduction in the frequency of administration of the OXM isachieved thanks to reverse pegylation which render the OXM more stableand more potent. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing T1/2 ofthe OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of reducing bloodclearance of OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing T1/2 ofthe OXM. In another embodiment, reduction in the frequency ofadministration of the OXM is achieved as a result of increasing the AUCmeasure of the OXM.

In another embodiment, a reverse pegylated OXM is administered to asubject once a day. In another embodiment, a reverse pegylated OXM isadministered to a subject once every two days. In another embodiment, areverse pegylated OXM is administered to a subject once every threedays. In another embodiment, a reverse pegylated OXM is administered toa subject once every four days. In another embodiment, a reversepegylated OXM is administered to a subject once every five days. Inanother embodiment, a reverse pegylated OXM is administered to a subjectonce every six days. In another embodiment, a reverse pegylated OXM isadministered to a subject once every week. In another embodiment, areverse pegylated OXM is administered to a subject once every 7-14 days.In another embodiment, a reverse pegylated OXM is administered to asubject once every 10-20 days. In another embodiment, a reversepegylated OXM is administered to a subject once every 5-15 days. Inanother embodiment, a reverse pegylated OXM is administered to a subjectonce every 15-30 days.

Oral administration, in one embodiment, comprises a unit dosage formcomprising tablets, capsules, lozenges, chewable tablets, suspensions,emulsions and the like. Such unit dosage forms comprise a safe andeffective amount of OXM of the invention, each of which is in oneembodiment, from about 0.7 or 3.5 mg to about 280 mg/70 kg, or inanother embodiment, about 0.5 or 10 mg to about 210 mg/70 kg. Thepharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for peroral administration are well-known in the art.In some embodiments, tablets typically comprise conventionalpharmaceutically-compatible adjuvants as inert diluents, such as calciumcarbonate, sodium carbonate, mannitol, lactose and cellulose; binderssuch as starch, gelatin and sucrose; disintegrants such as starch,alginic acid and croscarmelose; lubricants such as magnesium stearate,stearic acid and talc. In one embodiment, glidants such as silicondioxide can be used to improve flow characteristics of thepowder-mixture. In one embodiment, coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, areuseful adjuvants for chewable tablets. Capsules typically comprise oneor more solid diluents disclosed above. In some embodiments, theselection of carrier components depends on secondary considerations liketaste, cost, and shelf stability, which are not critical for thepurposes of this invention, and can be readily made by a person skilledin the art.

In one embodiment, the oral dosage form comprises predefined releaseprofile. In one embodiment, the oral dosage form of the presentinvention comprises an extended release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises a slow release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form of the presentinvention comprises an immediate release tablets, capsules, lozenges orchewable tablets. In one embodiment, the oral dosage form is formulatedaccording to the desired release profile of the long-acting OXN as knownto one skilled in the art.

In another embodiment, compositions for use in the methods of thisinvention comprise solutions or emulsions, which in another embodimentare aqueous solutions or emulsions comprising a safe and effectiveamount of the compounds of the present invention and optionally, othercompounds, intended for topical intranasal administration. In someembodiments, the compositions comprise from about 0.001% to about 10.0%w/v of a subject compound, more preferably from about 00.1% to about2.0, which is used for systemic delivery of the compounds by theintranasal route.

In another embodiment, the pharmaceutical compositions are administeredby intravenous, intra-arterial, subcutaneous or intramuscular injectionof a liquid preparation. In another embodiment, liquid formulationsinclude solutions, suspensions, dispersions, emulsions, oils and thelike. In one embodiment, the pharmaceutical compositions areadministered intravenously, and are thus formulated in a form suitablefor intravenous administration. In another embodiment, thepharmaceutical compositions are administered intra-arterially, and arethus formulated in a form suitable for intra-arterial administration. Inanother embodiment, the pharmaceutical compositions are administeredintramuscularly, and are thus formulated in a form suitable forintramuscular administration.

Further, in another embodiment, the pharmaceutical compositions areadministered topically to body surfaces, and are thus formulated in aform suitable for topical administration. Suitable topical formulationsinclude gels, ointments, creams, lotions, drops and the like. Fortopical administration, the compounds of the present invention arecombined with an additional appropriate therapeutic agent or agents,prepared and applied as solutions, suspensions, or emulsions in aphysiologically acceptable diluent with or without a pharmaceuticalcarrier.

In one embodiment, pharmaceutical compositions of the present inventionare manufactured by processes well known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

In one embodiment, pharmaceutical compositions for use in accordancewith the present invention is formulated in conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries, which facilitate processing of OXM into preparationswhich, can be used pharmaceutically. In one embodiment, formulation isdependent upon the route of administration chosen.

In one embodiment, injectables, of the invention are formulated inaqueous solutions. In one embodiment, injectables, of the invention areformulated in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological salt buffer. In someembodiments, for transmucosal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art.

In one embodiment, the preparations described herein are formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. In another embodiment, formulations for injection arepresented in unit dosage form, e.g., in ampoules or in multidosecontainers with optionally, an added preservative. In anotherembodiment, compositions are suspensions, solutions or emulsions in oilyor aqueous vehicles, and contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

The compositions also comprise, in another embodiment, preservatives,such as benzalkonium chloride and thimerosal and the like; chelatingagents, such as edetate sodium and others; buffers such as phosphate,citrate and acetate; tonicity agents such as sodium chloride, potassiumchloride, glycerin, mannitol and others; antioxidants such as ascorbicacid, acetylcystine, sodium metabisulfote and others; aromatic agents;viscosity adjustors, such as polymers, including cellulose andderivatives thereof; and polyvinyl alcohol and acid and bases to adjustthe pH of these aqueous compositions as needed. The compositions alsocomprise, in some embodiments, local anesthetics or other actives. Thecompositions can be used as sprays, mists, drops, and the like.

In one embodiment, pharmaceutical compositions for parenteraladministration include aqueous solutions of the active preparation inwater-soluble form. Additionally, suspensions of long acting OXM, insome embodiments, are prepared as appropriate oily or water basedinjection suspensions. Suitable lipophilic solvents or vehicles include,in some embodiments, fatty oils such as sesame oil, or synthetic fattyacid esters such as ethyl oleate, triglycerides or liposomes. Aqueousinjection suspensions contain, in some embodiments, substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In another embodiment, the suspensionalso contain suitable stabilizers or agents which increase thesolubility of long acting OXM to allow for the preparation of highlyconcentrated solutions.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In another embodiment, the pharmaceutical composition delivered in acontrolled release system is formulated for intravenous infusion,implantable osmotic pump, transdermal patch, liposomes, or other modesof administration. In one embodiment, a pump is used (see Langer, supra;Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).In another embodiment, polymeric materials can be used. In yet anotherembodiment, a controlled release system can be placed in proximity tothe therapeutic target, i.e., the brain, thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990).

In one embodiment, long acting OXM is in powder form for constitutionwith a suitable vehicle, e.g., sterile, pyrogen-free water basedsolution, before use. Compositions are formulated, in some embodiments,for atomization and inhalation administration. In another embodiment,compositions are contained in a container with attached atomizing means.

In one embodiment, the preparation of the present invention isformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

In one embodiment, pharmaceutical compositions suitable for use incontext of the present invention include compositions wherein longacting OXM is contained in an amount effective to achieve the intendedpurpose. In another embodiments, a therapeutically effective amountmeans an amount of long acting OXM effective to prevent, alleviate orameliorate symptoms of disease or prolong the survival of the subjectbeing treated.

In one embodiment, determination of a therapeutically effective amountis well within the capability of those skilled in the art.

The compositions also comprise preservatives, such as benzalkoniumchloride and thimerosal and the like; chelating agents, such as edetatesodium and others; buffers such as phosphate, citrate and acetate;tonicity agents such as sodium chloride, potassium chloride, glycerin,mannitol and others; antioxidants such as ascorbic acid, acetylcystine,sodium metabisulfote and others; aromatic agents; viscosity adjustors,such as polymers, including cellulose and derivatives thereof; andpolyvinyl alcohol and acid and bases to adjust the pH of these aqueouscompositions as needed. The compositions also comprise local anestheticsor other actives. The compositions can be used as sprays, mists, drops,and the like.

Some examples of substances which can serve aspharmaceutically-acceptable carriers or components thereof are sugars,such as lactose, glucose and sucrose; starches, such as corn starch andpotato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; vegetable oils, such as peanutoil, cottonseed oil, sesame oil, olive oil, corn oil and oil oftheobroma; polyols such as propylene glycol, glycerine, sorbitol,mannitol, and polyethylene glycol; alginic acid; emulsifiers, such asthe Tween™ brand emulsifiers; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents,stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions. The choice of apharmaceutically-acceptable carrier to be used in conjunction with thecompound is basically determined by the way the compound is to beadministered. If the subject compound is to be injected, in oneembodiment, the pharmaceutically-acceptable carrier is sterile,physiological saline, with a blood-compatible suspending agent, the pHof which has been adjusted to about 7.4.

In addition, the compositions further comprise binders (e.g. acacia,cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropylcellulose, hydroxypropyl methyl cellulose, povidone), disintegratingagents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide,croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate),buffers (e.g., Tris-HCI, acetate, phosphate) of various pH and ionicstrength, additives such as albumin or gelatin to prevent absorption tosurfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acidsalts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate),permeation enhancers, solubilizing agents (e.g., glycerol, polyethyleneglycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite,butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose,hydroxypropylmethyl cellulose), viscosity increasing agents (e.g.carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g. aspartame, citric acid), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid,magnesium stearate, polyethylene glycol, sodium lauryl sulfate),flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethylphthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropylcellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers orpoloxamines), coating and film forming agents (e.g. ethyl cellulose,acrylates, polymethacrylates) and/or adjuvants.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. For a suspension, typicalsuspending agents include methyl cellulose, sodium carboxymethylcellulose, cellulose (e.g. Avicel™, RC-591), tragacanth and sodiumalginate; typical wetting agents include lecithin and polyethylene oxidesorbitan (e.g. polysorbate 80). Typical preservatives include methylparaben and sodium benzoate. In another embodiment, peroral liquidcompositions also contain one or more components such as sweeteners,flavoring agents and colorants disclosed above.

The compositions also include incorporation of the active material intoor onto particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts.) Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance.

Also comprehended by the invention are particulate compositions coatedwith polymers (e.g. poloxamers or poloxamines) and the compound coupledto antibodies directed against tissue-specific receptors, ligands orantigens or coupled to ligands of tissue-specific receptors.

In one embodiment, compounds modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. Inanother embodiment, the modified compounds exhibit substantially longerhalf-lives in blood following intravenous injection than do thecorresponding unmodified compounds. In one embodiment, modificationsalso increase the compound's solubility in aqueous solution, eliminateaggregation, enhance the physical and chemical stability of thecompound, and greatly reduce the immunogenicity and reactivity of thecompound. In another embodiment, the desired in vivo biological activityis achieved by the administration of such polymer-compound abducts lessfrequently or in lower doses than with the unmodified compound.

In another embodiment, preparation of effective amount or dose can beestimated initially from in vitro assays. In one embodiment, a dose canbe formulated in animal models and such information can be used to moreaccurately determine useful doses in humans.

In one embodiment, toxicity and therapeutic efficacy of the long actingOXM as described herein can be determined by standard pharmaceuticalprocedures in vitro, in cell cultures or experimental animals. In oneembodiment, the data obtained from these in vitro and cell cultureassays and animal studies can be used in formulating a range of dosagefor use in human. In one embodiment, the dosages vary depending upon thedosage form employed and the route of administration utilized. In oneembodiment, the exact formulation, route of administration and dosagecan be chosen by the individual physician in view of the patient'scondition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1].

In one embodiment, depending on the severity and responsiveness of thecondition to be treated, dosing can be of a single or a plurality ofadministrations, with course of treatment lasting from several days toseveral weeks or until cure is effected or diminution of the diseasestate is achieved.

In one embodiment, the amount of a composition to be administered will,of course, be dependent on the subject being treated, the severity ofthe affliction, the manner of administration, the judgment of theprescribing physician, etc.

In one embodiment, compositions including the preparation of the presentinvention formulated in a compatible pharmaceutical carrier are also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

In another embodiment, a reverse pegylated OXM as described herein isadministered via systemic administration. In another embodiment, areverse pegylated OXM as described herein is administered byintravenous, intramuscular or subcutaneous injection. In anotherembodiment, a reverse pegylated OXM as described herein is lyophilized(i.e., freeze-dried) preparation in combination with complex organicexcipients and stabilizers such as nonionic surface active agents (i.e.,surfactants), various sugars, organic polyols and/or human serumalbumin. In another embodiment, a pharmaceutical composition comprises alyophilized reverse pegylated OXM as described in sterile water forinjection. In another embodiment, a pharmaceutical composition comprisesa lyophilized reverse pegylated OXM as described in sterile PBS forinjection. In another embodiment, a pharmaceutical composition comprisesa lyophilized reverse pegylated OXM as described in sterile 0.9% NaClfor injection.

In another embodiment, the pharmaceutical composition comprises areverse pegylated OXM as described herein and complex carriers such ashuman serum albumin, polyols, sugars, and anionic surface activestabilizing agents. See, for example, WO 89/10756 (Hara etal.—containing polyol and p-hydroxybenzoate). In another embodiment, thepharmaceutical composition comprises a reverse pegylated OXM asdescribed herein and lactobionic acid and an acetate/glycine buffer. Inanother embodiment, the pharmaceutical composition comprises a reversepegylated OXM as described herein and amino acids, such as arginine orglutamate that increase the solubility of interferon compositions inwater. In another embodiment, the pharmaceutical composition comprises alyophilized reverse pegylated OXM as described herein and glycine orhuman serum albumin (HSA), a buffer (eg. acetate) and an isotonic agent(e.g NaCl). In another embodiment, the pharmaceutical compositioncomprises a lyophilized reverse pegylated OXM as described herein andphosphate buffer, glycine and HSA.

In another embodiment, the pharmaceutical composition comprising apegylated or reverse pegylated OXM as described herein is stabilizedwhen placed in buffered solutions having a pH between about 4 and 7.2.In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is stabilized with an aminoacid as a stabilizing agent and in some cases a salt (if the amino aciddoes not contain a charged side chain).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is a liquid compositioncomprising a stabilizing agent at between about 0.3% and 5% by weightwhich is an amino acid.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein provides dosing accuracy andproduct safety. In another embodiment, the pharmaceutical compositioncomprising a reverse pegylated OXM as described herein provides abiologically active, stable liquid formulation for use in injectableapplications. In another embodiment, the pharmaceutical compositioncomprises a non-lyophilized reverse pegylated OXM as described herein.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein provides a liquid formulationpermitting storage for a long period of time in a liquid statefacilitating storage and shipping prior to administration.

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein comprises solid lipids asmatrix material. In another embodiment, the injectable pharmaceuticalcomposition comprising a reverse pegylated OXM as described hereincomprises solid lipids as matrix material. In another embodiment, theproduction of lipid microparticles by spray congealing was described bySpeiser (Speiser and al., Pharm. Res. 8 (1991) 47-54) followed by lipidnanopellets for peroral administration (Speiser E P 0167825 (1990)). Inanother embodiment, lipids, which are used, are well tolerated by thebody (e.g. glycerides composed of fatty acids which are present in theemulsions for parenteral nutrition).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein is in the form of liposomes(J. E. Diederichs and al., Pharm./nd. 56 (1994) 267-275).

In another embodiment, the pharmaceutical composition comprising areverse pegylated OXM as described herein comprises polymericmicroparticles. In another embodiment, the injectable pharmaceuticalcomposition comprising a reverse pegylated OXM as described hereincomprises polymeric microparticles. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises nanoparticles. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises liposomes. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid emulsion. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises microspheres. In another embodiment, thepharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid nanoparticles. In another embodiment,the pharmaceutical composition comprising a reverse pegylated OXM asdescribed herein comprises lipid nanoparticles comprising amphiphiliclipids. In another embodiment, the pharmaceutical composition comprisinga reverse pegylated OXM as described herein comprises lipidnanoparticles comprising a drug, a lipid matrix and a surfactant. Inanother embodiment, the lipid matrix has a monoglyceride content whichis at least 50% w/w.

In one embodiment, compositions of the present invention are presentedin a pack or dispenser device, such as an FDA approved kit, whichcontain one or more unit dosage forms containing the long acting OXM. Inone embodiment, the pack, for example, comprise metal or plastic foil,such as a blister pack. In one embodiment, the pack or dispenser deviceis accompanied by instructions for administration. In one embodiment,the pack or dispenser is accommodated by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions or human orveterinary administration. Such notice, in one embodiment, is labelingapproved by the U.S. Food and Drug Administration for prescription drugsor of an approved product insert.

In one embodiment, it will be appreciated that the reverse pegylated OXMof the present invention can be provided to the individual withadditional active agents to achieve an improved therapeutic effect ascompared to treatment with each agent by itself. In another embodiment,measures (e.g., dosing and selection of the complementary agent) aretaken to adverse side effects which are associated with combinationtherapies.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference. Other general references are provided throughout thisdocument.

Materials and Methods

PEG30-FMS-OXM Synthesis—Heterogeneous

1.1 Stage 1: OXM Synthesis

Oxyntomodulin was synthesized which consists of the following peptidesequence:

HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO: 1)

The peptide was synthesized by the solid phase method employing theFmoc-strategy throughout the peptide chain assembly (Almac Sciences,Scotland).

The peptide sequence was assembled using the following steps:

1. Capping

The resin was capped using 0.5M acetic anhydride (Fluka) solution in DMF(Rathburn).

2. Deprotection

Fmoc-protecting group was removed from the growing peptide chain using20% v/v piperidine (Rathburn) solution in DMF (Rathburn).

3. Amino Acid Coupling

0.5M Amino acid (Novabiochem) solution in DMF (Rathburn) was activatedusing 1M HOBt (Carbosynth) solution in DMF (Rathburn) and 1M DIC(Carbosynth) solution in DMF (Rathburn). 4 equivalents of each aminoacid were used per coupling.

The crude peptide is cleaved from the resin and protecting groupsremoved by stirring in a cocktail of Triisopropylsilane (Fluka), water,dimethylsulphide (Aldrich), ammonium iodide (Aldrich) and TFA (AppliedBiosystems) for 4 hours. The crude peptide is collected by precipitationfrom cold diethyl ether.

Peptide Purification

Crude peptide was dissolved in acetonitrile (Rathburn)/water (MilliQ)(5:95) and loaded onto the preparative HPLC column. The chromatographicparameters are as follows:

Column: Phenomenex Luna C18 250 mm×30, 15 μm, 300A

Mobile Phase A: water+0.1% v/v TFA (Applied Biosystems)

Mobile Phase B: acetonitrile (Rathburn)+0.1% v/v TFA (AppliedBiosystems)

UV Detection: 214 or 220 nm

Gradient: 25% B to 31% B over 4 column volumes

Flow rate 43 mL/min

Stage 2—Linker Synthesis

The synthesis of compounds 2-5 is based on the procedures described byAlbericio et al. in Synthetic Communication, 2001, 31(2), 225-232.

2-(Boc-amino)fluorene (2):

2-Aminofluorene (18 g, 99 mmol) was suspended in a mixture ofdioxane:water (2:1) (200 ml) and 2N NaOH (60 ml) in an ice bath withmagnetic stirring. Boc₂O (109 mmol, 1.1 eq) was then added and stirringcontinued at RT. The reaction was monitored by TLC (Rf=0.5, Hex./EthylAcetate 2:1) and the pH maintained between 9-10 by addition of 2N NaOH.At reaction completion, the suspension was acidified with 1M KHSO4 topH=3. The solid was filtered and washed with cold water (50 ml),dioxane-water (2:1) and then azeotroped with toluene twice before usingit in the next step.

9-Formyl-2-(Boc-amino)fluorene (3):

In a 3 necked RBF, NaH (60% in oil; 330 mmol, 3.3 eq) was suspended indry THF (50 ml), a solution of -(Boc-amino)fluorine described in step 2(28 g; 100 mmol) in dry THF (230 ml) was added dropwise over 20 minutes.A thick yellow slurry was observed and the mixture stirred for 10minutes at RT under nitrogen. Ethyl formate (20.1 ml, 250 mmol, 2.5 eq)was added dropwise (Caution: gas evolution). The slurry turned to a palebrown solution. The solution was stirred for 20 minutes. The reactionwas monitored by TLC (Rf=0.5, Hex./Ethyl acetate 1:1) and when onlytraces of starting material was observed, it was quenched with icedwater (300 ml). The mixture was evaporated under reduce pressure untilmost of the THF has been removed. The resulting mixture was treated withacetic acid to pH=5. The white precipitate obtained was dissolved inethyl acetate and the organic layer separated. The aqueous layer wasextracted with ethyl acetate and all the organic layer combined andwashed with saturated sodium bicarbonate, brine and dried over MgSO₄.After filtration and solvent removal a yellow solid was obtained. Thismaterial was used in the next step.

9-Hydroxymethyl-2-(Boc-amino)fluorene (4):

Compound 3 from above was suspended in MeOH (200 ml) and sodiumborohydride was added portion wise over 15 minutes. The mixture wasstirred for 30 minutes (caution: exothermic reaction and gas evolution).The reaction was monitored by TLC (Rf=0.5, Hex./EtOAc 1:1) and wascompleted. Water (500 ml) was added and the pH adjusted to 5 with aceticacid. The work up involved extraction twice with ethyl acetate, washingthe combined organic layers with sodium bicarbonate and brine, dryingover MgSO₄, filtration and concentration to dryness. The crude obtainedwas purified by flask chromatography using Heptane/EtOAc (3:1) to give ayellow foam (36 g, 97.5% purity, traces of ethyl acetate and diethylether observed in the 1H-NMR).

MAL-Fmoc-NHS (7):

To a clean dry 500 ml RBF with overhead agitation was chargedtriphosgene (1.58 g, 0.35 eq.) in dry THF (55 ml) to form a solution atambient. This was cooled to 0° C. with an ice/water bath and a solutionof NHS (0.67 g, 0.38 eq) in dry THF (19 ml) added dropwise over 10minutes under nitrogen at 0° C. The resultant solution was stirred for30 minutes. A further portion of NHS (1.34 g, 0.77 eq) in dry THF (36ml) was added dropwise at 0° C. over 10 minutes and stirred for 15minutes.

Compound 6 (5.5 g, 1 eq), dry THF (55 ml) and pyridine (3.07 ml, 2.5 eq)were stirred together to form a suspension. This was added to the NHSsolution in portions a 0-5° C. and then allowed to go to RT by removingthe ice bath.

After 20 hours the reaction was stop (starting material still present,if the reaction is pushed to completion a dimmer impurity has beenobserved).

The reaction mixture was filtered and to the filtrate, 4% brine (200 ml)and EtOAc (200 ml) were added. After separation, the organic layer waswashed with 5% citric acid (220 ml) and water (220 ml). The organiclayer was then concentrated to give 7.67 g of crude MAL-Fmoc-NHS. Thematerial was purified by column chromatography using a gradientcyclohexane/EtOAc 70:30 to 40:60. The fractions containing product wereconcentrated under vacuum to give 3.47 g (45%) of MAL-Fmoc-NHS.

MAL-FMS-NHS:

To a solution of MAL-Fmoc-NHS (100 mg, 0.2 mmol) in trifluoroacetic acid(10 ml), chlorosulfonic acid (0.5 ml) was added. After 15 minutes,ice-cold diethyl ether (90 ml) was added and the product precipitated.The material was collected by centrifugation, washed with diethyl etherand dried under vacuum. 41.3 mg (35%) of beige solid was obtained.

Stage 3—Conjugation

Heterogenous conjugation of the 3 amines sites in the OXM peptide(Lys12, Lys30 and amino terminal) performed as “one pot reaction” inwhich 1 eq from each component: OXM, mPEG-SH and FMS linker as mixedtogether at Ph 7.2 for 30 min. The reaction stopped by adding aceticacid to reduce PH to 4.

PEG30-FMS-OXM Synthesis—Homogeneous

Stage 1: The Spacer

MAL-FMS-NHS (FMS) synthesis: as described for the heterogenousconjugate.

Stage 2: Oxyntomodulin (OXM) Synthesis:

N-terminus site directed OXM—as described above for heterogeneousconjugate.

Lys₁₂ or Lys₃₀ site directed OXM—Using the same strategy except forusing Fmoc-Lys(ivDde)-OH in position 12 or 30 of lysine andBoc-His(Boc)-OH as the last amino acid to be coupled.

Stage 3: Homogeneous Conjugation

Coupling FMS to OXM:

MAL-FMS-NHS linker solution (0.746 ml, 10 mg/ml in DMF, 2 eq) was addedto OXM resin (1 eq, 200 mg resin, 31.998 μmol/g free amine) DMF wasadded until resin was just freely mobile and then sonicated for 19 hrs.Resin was washed with DMF and Methanol before drying overnight in vacuumdesiccator. The cleavage cocktail contained TFA/TIS/H2O. The cleavagewas performed over 3.5 hrs at room temperature. After filtration of theresin, the FMS-OXM was precipitated in cold diethyl ether. 42.1 mg ofcrude FMS-OXM (36% pure) was obtained at the end of the cleavage stage.

Coupling FMS to Lys₁₂ Site Directed OXM:

MAL-FMS-NHS linker solution (10 mg/ml in DMF, 2.5 equiv.) was added to(Lys12)OXM resin (1 equiv.) with addition of DIEA (5 equiv.). DMF wasadded until resin was just freely mobile and then sonicated overnight.Resin was washed with DMF and Methanol before drying overnight in vacuumdesiccator. Cleavage and precipitation as described for N-terminal sitedirected.

Coupling FMS to Lys₃₀ Site Directed OXM:

MAL-FMS-NHS linker (2.5 equiv.) was solubilized in DCM with addition ofDIEA (5 equiv.). This linker/DIEA solution was added to (Lys30)OXM resinthen sonicated overnight. Resin was washed with DCM and Methanol beforedrying overnight in vacuum desiccator. Cleavage and precipitation asdescribed for N-terminal site directed.

Purification

The resultant crude FMS-OXM was purified in one portion.

Sample diluent: 10% Acetonitrile in water

Column: Luna C18 (2), 100 Å, 250×21.2 mm

Injection flow rate: 9 ml/min

Run flow rate: 9 ml/min

Buffer A: Water (0.1% TFA)

Buffer B: Acetonitrile (0.1% TFA)

Gradient: 10-45% B over 32 mins

Monitoring: 230 nm

Conjugation of PEG30 to FMS-OXM

FMS-OXM solution (1 equiv, 15.1 mg in 1.5 ml DMF) was prepared. PEG30 (1equiv, 9.2 ml of 10 mg/ml in pH 6.5 phosphate buffer) was added to theFMS-OXM solution. The reaction mixture was then stirred for 30 mins atroom temperature before adding glacial acetic acid (200 μl) to quenchreaction by lowering the pH.

The resultant reaction mixture was then purified using RP-HPLC.

Column: Luna C18 (2), 100 Å, 250×21.2 mm

Injection flow rate: 5 ml/min

Run flow rate: 20 ml/min

Buffer A: Water & 0.1% TFA

Buffer B: Acetonitrile/Water (75:25) & 0.1% TFA

Gradient: 10-65% B over 41 mins

Monitoring: 220, 240, 280 nm

IP Glucose Tolerance Test

C57BL/6 male mice were fasted overnight then weighed, and blood glucoselevels were measured by tail vein sampling using a handheld glucometer.Mice were IP injected with PEG-SH (vehicle), PEG30-FMS-OXM(Heterogeneous) and the three homogeneous variants of PEG30-FMS-OXM(amino, Lys12 and Lys30). Glucose (1.5 gr/kg) was administrated IP 15min after test article administration. Blood glucose levels weremeasured by tail vein sampling at prior to glucose administration and10, 20, 30, 60, 90, 120 and 180 min after glucose administration using ahandheld glucometer.

In-Vitro Characterization of GLP-1 Receptors Activation

Activation of GLP-1 receptor was assessed using two different celllines; HTS163C2 (Millipore) and cAMP Hunter™ CHO-K1 GLP1R (Discoverx),both are over expressing the GLP-1 receptor. The HTS163C2 (Millipore)were seeded in 96 wells half-area white plate (Greiner) at a density of100,000 cells/ml and incubated for 24 hours at 37° C. The cells wereincubated with escalating concentrations of heterogeneous PEG30-FMS-OXMand 3 homogeneous PEG30-FMS-OXM variants (amino, Lys12 and Lys30). CellscAMP concentrations were quantified by HTRF assay (Cisbio 62AM4PEB) andEC50 parameter was analyzed by PRISM software. The cAMP Hunter™ CHO-K1GLP1R secretes cAMP upon binding of the ligand to the receptor. Cells ata density of 500000 cells/ml were seeded in 96 wells plate, and wereincubated for 24 h at 37° C. with 5% CO₂ Ligands were diluted in diluentcontains IBMX and were added in duplicate to the culture wells for 30min at 37° C. with 5% CO₂. The concentration range of PEG30-FMS-OXM was1.5*10⁻¹⁰ to 1.2*10⁻⁶ M. Lysis buffer and detector reagents were addedto the wells and cAMP concentrations were detected using achemiluminescent signal. The dose dependent curves were established andthe binding affinities (EC50) of various ligands were calculated usingPRISM software by applying the best fit dose response model (Fourparameters).

In-Vitro Characterization of Glucagon Receptors Activation

Activation of glucagon receptor was assessed using cAMP Hunter™ CHO-K1GCGR cell-line that over expresses glucagon-receptor. This cell-linesecretes cAMP upon binding of the ligand to the glucagon receptor. Cellswere seeded at a density of 500000 cells/ml in 96 wells plate, and wereincubated for 24 h at 37° C. with 5% CO₂ Ligands were diluted in diluentcontains IBMX and were added in duplicate to the culture wells for 30min at 37° C. with 5% CO₂. The concentration range of MOD-6031 was5.8*10⁻¹¹ to 2.7*10⁻⁷ M. Lysis buffer and detector reagents were addedto the wells and cAMP concentrations were detected using achemiluminescent signal. The dose dependent curves were established andthe binding affinities (EC50) of various ligands were calculated usingPRISM software by applying the best fit dose response model (Fourparameters).

Obese (Ob/Ob) Mice Model

Study 1: Twenty five male ob/ob mice (male, B6. V-Lep{circumflex over( )}ob/OlaHsd, 5-6 weeks of age, Harlan) were acclimatized to thefacility (10 days) followed by handling protocol whereby animals werehandled as if to be dosed but were actually not weighed or dosed (10days). Subsequently, animals underwent baseline period for 7 days inwhich they were dosed twice a week with the appropriate vehicle by thesubcutaneous route in volume of 20 ml/kg. Body weight, food and waterintake were recorded daily, and samples were taken for non-fasting andfasting glucose measurements and non-fasting and fasting insulinmeasurements. Animals were subsequently allocated into five treatmentgroups (N=5) based on body weight and glycemic profile. Animals weredosed every four days (days: 1, 5, 9, 13 and 16) as describes intable 1. During the treatment period, food intake, water intake and bodyweight have been measured and recorded daily, before dosing. Severalprocedures and sampling have been performed: non-fasting and fastingglucose on days 2, 6, 14 and 17 (on day 17 only non-fasting glucose wasmeasured), fasting and non-fasting insulin (days 2, 6 and 14). Terminalsamples on day 19 were analyzed for cholesterol.

TABLE 1 Study design Group Treatment (sc) Frequency n 1 PEG-SH (142.86mg/ml) Days 1, 5, 9, 13 and 16 5 2 PEG-FMS-OXM Hetero (MOD- Days 1, 5,9, 13 and 16 5 6030). 2000 nmol/kg 3 Amino PEG-FMS-OXM Days 1, 5, 9, 13and 16 5 2000 nmol/kg 4 Lys12 PEG-FMS-OXM Days 1, 5, 9, 13 and 16 5 2000nmol/kg 5 Lys30 PEG-FMS-OXM Days 1, 5, 9, 13 and 16 5 2000 nmol/kg

Study 2:

One hundred male ob/ob mice (5-6 weeks of age, Charles River) wereacclimatized to the facility (3 days) followed by handling protocolwhereby animals were handled as if to be dosed but were actually notweighed or dosed (7 days). Subsequently, animals were underwent baselineperiod for 7 days in which they were dosed twice a week with PEG30-SHvehicle (146 mg/ml) by a subcutaneous route in volume of 20 ml/kg. Bodyweight, food and water intake were recorded daily. Subsequently animalswere allocated into 8 treatment, control and pair fed groups (groupsA-H, N=8) (table 2). The pair fed group was pair-fed to the high dose(6000 nmol/kg) group of MOD-6031 and it was given the daily food rationequal to that eaten by its paired counterpart in group D the previousday. 3 additional groups (groups I-K, N=12) were administered withMOD-6031 at 1000, 3000 and 6000 nmol/kg and were used for sampling forPK analysis. PEG-SH vehicle (292 mg/ml), MOD-6031 at 1000, 3000 and 6000nmol/kg, and the pair fed groups were administered twice a week for 32days while OXM, Liraglutide® and PBS were administered bid. Body weight,food and water intake were measured daily. Non-fasting and fastingglucose were measured once a week, OGTT were performed on days 2 and 30.Terminal blood samples (day 33) were analyzed for glucose, insulin,Cholesterol, and MOD-6031, PEG-FMS and OXM concentrations. Mice in thePK groups received a single dose of MOD-6031 and blood samples weretaken at 4, 8, 24, 36, 48, 72, 96 and 120 h (n=3 per time point) for PKanalysis allows to quantify MOD-6031 and its compounds concentrations byLC-MS/MS method.

TABLE 2 Study design Group Treatment (sc) n Frequency A PEG30-SH Vehicle(292 mg/kg; 8 Twice a week on 20 ml/kg) days 1, 4, 8, B MOD-6031 1000nmoles/kg 8 11, 15, 18, 22, C MOD-6031 3000 nmoles/kg 8 25, 29 and 32 DMOD-6031 6000 nmoles/kg 8 E PEG30-SH Vehicle (292 mg/kg) 8 Pair-Fed toGroup D F PBS bid (10 ml/kg) 8 b.i.d for 32 days G OXM 6000 nmoles/kgbid (10 ml/kg) 8 H Liraglutide 0.1 mg/kg bid (10 ml/kg) 8 I MOD-60311000 nmoles/kg PK group 12 Single injection J MOD-6031 3000 nmoles/kg PKgroup 12 on day 1 K MOD-6031 6000 nmoles/kg PK group 12

Results Example 1 Manufacturing and Development Synthesis

The composition of the PEG-FMS-OXM conjugate depends on its synthesisprocedure. Different variants of the PEG-FMS-OXM conjugate were produced(FIG. 1).

Heterogenous Conjugate:

Synthesis of MOD-6030 (PEG₃₀-FMS-OXM) was performed as follows: FMSspacer was mixed with OXM and PEG(30)-SH (as one pot reaction). The FMSspacer was coupled to OXM by its NHS activated ester on one side and byPEG-SH connected to the maleimide group on the other sidesimultaneously. This way, a heterogeneous mixture of PEG-FMS-OXMconjugate is composed of three variants connected by one of the 3 aminesof the OXM peptide (N-terminal, Lys₁₂ and Lys₃₀).

Homogeneous Conjugate:

The conjugation procedure was further developed into a two steps processin which attachment to the FMS spacer was executed in a controlled andsite directed manner. In the first step, the FMS spacer was coupled tothe OXM (on resin partially protected OXM), then cleaved followed byde-protection and purification of FMS-OXM (by RP-HPLC). The second stepwas the attachment of PEG30-SH to the purified homogeneous FMS-OXM. Thefinal conjugated product is further purified by RP-HPLC. Additionalpurification steps may be applied such as Ion exchange or SEC-HPLC orany other purification step.

Three peptides on resin were synthesized using Fmoc solid phasestrategy. For synthesis of the homogeneous conjugate connected by aminoacid lysine in position 12 or 30 of the OXM, a selective protectinggroup was applied for either Lys12 or Lys30 of OXM as ivDde(1-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidine)ethyl]), which can beremoved under basic conditions while the rest of the peptide is still onthe resin with the other protective groups.

Therefore, three resin-bound OXMs were synthesized: N-terminal—usingprotection groups suitable for solid phase synthesis with Fmoc strategy(usually Boc protecting group is used for the ε amine) and Lys₁₂ orLys₃₀ with ivDde protection group. These OXM peptides were intended forfurther selective coupling with the FMS linker.

Homogenous conjugates performed as ‘on resin synthesis’. The conjugatesynthesized in two steps: 1. Coupling between the OXM and FMS, cleavageand purification 2. Pegylation of OXM-FMS with PEG₃₀-SH. In thisprocedure, the coupling of the FMS linker is done with the OXM, while itis bound to the resin. The OXM was fully protected, allowing thespecific un-protected desired amino site on OXM to react with the NHSmoiety. The purified FMS-OXM was attached to the PEG-SH. The crudeconjugate was purified using HPLC (RP or Cation exchange or both).

Example 2 In-Vitro Characterization of GLP-1 Receptor Activation

GLP-1 receptor binding activation of PEG-FMS-OXM (MOD-6030;heterogenous) and 3 different homogeneous variants of PEG-FMS-OXM; theamino (MOD-6031), Lys12 and Lys30 were assessed using two differentcell-lines over expressing GLP-1 receptor; the Millipore HTS163C2 cellline and the cAMP Hunter™ CHO-K1 GLP1R. The potencies were determined bycalculating the EC50 of each variant, followed by calculating therelative potency of each variant to the heterogeneous (MOD-6030) version(dividing EC50 of each homogenous variant by the EC50 of theheterogeneous version and multiplying it by 100). The EC50 values andcalculated relative potencies are presented in table 3. For comparison,the binding affinity of OXM and GLP-1 to GLP-1 receptor of cAMP HunterCHO-K1 GLP1R cell line were measured.

TABLE 3 GLP-1 and Glucagon receptors binding activation cAMP Hunter ™cAMP Hunter ™ Millipore HTS163C2 CHO-K1 GLP1R CHO-K1 GCGR RelativeRelative Relative potency to potency to potency to EC50 heterogeneousEC50 heterogeneous EC50 heterogeneous (nM) (%) (nM) (%) (nM) (%) HeteroPEG₃₀- 76.2 100 8.14 ± 1.35 100 11.32 ± 3.26 100 FMS-OXM PEG₃₀-FMS- 55.272.24 8.07 ± 0.21 99.1 10.31 ± 2.87 91.1 OXM AMINO PEG₃₀-FMS- 179 234.99.42 ± 1.77 115.7 20.21 ± 4.12 178.5 OXM Lys₁₂ PEG₃₀-FMS- 307 402.917.34 ± 2.37  213.0  6.12 ± 1.75 54.1 OXM Lys₃₀ Oxyntomodulin 1.38 ±0.68  1.02 ± 0.32 (OXM) GLP-1 0.016 ± 0.006 NA Glucagon NA  0.04 ± 0.011

The relative potencies of the homogeneous variants were compared to theheterogeneous version and summarized in Table 3. Comparable bioactivityof the amino variant and the heterogeneous variant exhibited a relativepotency of 72.2% and 99.1% measured using the Millipore HTS163C2 and thecAMP Hunter™ CHO-K1 GLP1R, respectively.

The Lys12 and Lys30 variants had shown 2 and 4 fold reduction of GLP-1receptor binding activation using the Millipore HTS163C2 cell line whileonly showing minor and a 2 fold reduction, respectively, using the cAMPHunter™ CHO-K1 GLP1R cell line. The fact the amino variant demonstratedsuperior binding activity compared to the other variants is unexpectedas the N-terminus of OXM was reported to be involved in the binding ofOXM to the GLP-1 receptor (Druce et al., 2008). Overall, comparablebioactivity was shown for the amino variant and the heterogeneousvariant. GLP-1 receptor binding activations of OXM and GLP-1 peptideswere measured. It was found that OXM and GLP-1 had shown higher receptorbinding activation by 5.9 and 508.7 fold compared to the heterogeneousPEG30-FMS-OXM.

Example 3 In-Vitro Characterization of Glucagon Receptor Activation

Binding affinities of PEG-FMS-OXM variants to the glucagon receptor weredetermined using cAMP Hunter™ CHO-K1 GCGR cell-line that over expressesglucagon-receptor. This cell line was used to characterize theheterogeneous PEG-FMS-OXM (MOD-6030) and 3 different homogeneousvariants of PEG-FMS-OXM; the amino (MOD-6031), Lys12 and Lys30. Thepotencies were determined by calculating the EC50 of each variant,followed by calculating the relative potency of each variant to theheterogeneous version (dividing EC50 of each homogenous variant by theEC50 of the heterogeneous version and multiplying the value by 100). TheEC50 values and calculated relative potencies are presented in table 3.Amino variant showed comparable binding activity to the heterogeneousversion. The Lys30 variant showed the highest bioactivity and Lys12 hadshown 1.8 fold reductions. Glucagon receptor binding activations of OXMand glucagon peptides were measured. It was found that OXM and glucagonhad shown higher receptor binding activation by 11.1 and 283 foldcompared to the heterogeneous PEG30-FMS-OXM.

Example 4 Induction of Glucose Tolerance by PEG30-FMS-OXM Variants

In order to evaluate the in vivo activity of the heterogeneousPEG₃₀-FMS-OXM and the three PEG₃₀-FMS-OXM variants (amino, Lys₁₂ andLys₃₀), the IPGTT model was applied. Overnight fasted C57BL/6 mice wereinjected IP with the different compounds and a vehicle (PEG-SH) followedby IP injection of glucose and measurement of blood glucose levels fromthe tail vein using a glucometer. PEG-SH (238.10 nmol/kg), heterogeneousand homogeneous PEG₃₀-FMS-OXM, 100 nmol/kg peptide content) wereadministered IP 15 min prior to glucose IP injection (1.5 gr/kg). Allthe compounds induced glucose tolerance compared to vehicle group.Surprisingly, the homogeneous amino variant was slightly less potentcompared to the two other variants and to the heterogeneousPEG₃₀-FMS-OXM (table 4 FIG. 3) reflected by the slightly higher glucoseAUC compared to other variants, as opposed to the in-vitro activityresults. Yet, all variants significantly improved glucose tolerance ascompared to the vehicle PEG-SH control.

TABLE 4 Glucose tolerance in C57BL/6 mice % AUC % AUC AUC from AUC from(−60-180) control (0-180) control PEG-SH 26857 100 22522 100Heterogeneous 18200 67.8 13541 60.1 PEG₃₀-FMS-OXM PEG30-FMS-OXM 1989174.1 15781 70.1 AMINO variant PEG30-FMS-OXM 17652 65.7 13953 62.0 Lys12variant PEG30-FMS-OXM 17818 66.3 13159 58.4 Lys30 variant

The heterogeneous and homogeneous variants of the reversiblePEG₃₀-FMS-OXM were shown to be active both in-vitro and in the IPGTTmodel in-vivo. Surprisingly, the in-vitro results were not aligned withwhat is suggested in the literature, that the N-terminus of native OXMis involved in the peptide binding to the GLP-1 receptor; therefore, itwas expected that the amino terminus variant would show the lowestpotency both in-vitro and in-vivo. However, the homogeneous aminovariant of PEG₃₀-FMS-OXM demonstrated improved GLP-1 receptor activationcompared to the two other homogeneous variants using two different celllines (table 3) while demonstrating comparable efficacy in the IPGTT invivo model. The IPGTT in vivo model seems to present comparable activity(considering the variability between the animals). Although differentin-vitro binding activates to the GLP-1R and the GCGR were observedbetween the different PEG30-FMS-OXM variants, comparable ability toinduce glucose tolerance was shown (table 3 and 4). Unexpectedly, thesuperior in vitro activity of homogeneous amino PEG₃₀-FMS-OXM as shownin the cAMP induction assay was not reflected in the in vivo IP glucosetolerance test. The homogeneous amino variants PEG₃₀-FMS-OXM showed thelowest glucose tolerance profile compared to the two other variants andto the heterogeneous PEG₃₀-FMS-OXM. However, it still showed significantglucose tolerance effect in comparison to the vehicle (FIG. 3).

Example 5 Improvement of Body Weight, Glycemic and Lipid Profiles byPEG30-FMS-OXM Variants in Ob/Ob Mice Model

The ob/ob mice model exhibits a mutation of the ob gene such that theycannot produce leptin and develop a phenotype characterized byhyperinsulinaemia, obesity, hyperphagia, insulin resistance andsubsequently hyperglycaemia. These mice were used as a genetic model ofdiabetes in two different studies in order to evaluate the efficacy ofPEG30-FMS-OXM (Heterogeneous) and the three homogeneous variants ofPEG30-FMS-OXM (amino, Lys12 and Lys30).

Study 1: This study compared the efficacy of homogeneous variants(amino, Lys12 and Lys30) and the heterogeneous MOD-6030 whenadministered at 2000 nmol/kg. Reductions of body weight were obtainedfor all tested articles compared to vehicle (PEG-SH) group with finalreduction (on day 18) of 3.1%, 4.7%, 4.9% and 6.5% for Lys12, MOD-6030,amino and Lys30 variants, respectively (FIG. 4). Body weight reductionswere observed following drug injection on days 1, 5, 13 and 16 (FIG. 4).Reduction of food intake was observed for all treated groups followingdrug administration (except day 9) (FIG. 5). Measurement of glycemicparameters along the study had shown improvement of non-fasting glucose(FIG. 6a ) for amino and Lys12 treated groups and improvement of fastingglucose for all treated groups (FIG. 6b ). All treated groups showedsignificantly lower level of insulin compared to the control. Of note,the administered dose in this study was 2000 nmol/kg which is the lowereffective dose of MOD-6030 and thus the improvement of body weight, foodintake and glycemic profile were relatively moderate. Unexpectedly theamino variant was the only variant which showed superior efficacies inthe ability to reduce weight, inhibit food intake and to improveglycemic control. From a manufacturing perspective, on resin synthesisof the amino variant is the most straight forward procedure consideringthat the peptide in solid phase synthesis is extended from the aminoterminus. The terminal amine has preferred availability for couplingthan the internal amine groups of the Lysine at positions 12 and 30.This accessibility is reflected in the higher manufacturing yields ofthe amino variant as compared to the Lys12 and Lys30 variants. Anadditional benefit is that the synthesis towards the amino variantremains unchanged relative to OXM synthesis for the heterogeneousvariant, while the synthesis of Lys12 and Lys30 variants was modified bychanging the Lys used for the peptide synthesis and by the addition of aselective cleavage step (selectively removing the protecting group ofthe Lys). The OXM synthesis as previously developed for theheterogeneous was already optimized to achieve better yield androbustness. Overall, from a manufacturing perspective, synthesis ofamino variant on-resin is straight forward and possesses an advantageover the alternative variants. Being a homogenous variant, it also hasan advantage over a heterogeneous variant in that it is more suitablefor drug development and drug treatment.

Study 2: This study investigated the chronic effect of twice a weekadministration of MOD-6031 (the amino variants) at 1000, 3000 and 6000nmol/kg, on pharmacological and pharmacokinetic parameters in ob/ob micemodel, while OXM and liraglutide (long-acting GLP-1 receptor agonist)were evaluated as reference compounds. The measured pharmacologicalparameters were body weight, food and water intake, glucose control andlipid profile. Twice a week administration of high dose of MOD-6031(6000 nmol/kg) significantly reduced food intake and body weight (FIGS.7, 8), while the lower doses (3000 and 1000 nmol/kg) had shown lowereffects. At the conclusion of the study (day 33) animals of 1000, 3000and 6000 nmol/kg had shown body weight reduction of 5.2%, 12.3% and28.3%, respectively. The pair fed group, which were paired to the highdose group and ate equal amount of food (except the fasting days), had abody weight reduction of 12.7% while undergoing similar food intake.This phenomenon can be attributed to the ability of the amino variant ofPEG30-FMS-OXM to increase energy expenditure and thus animals that weretreated with 6000 nmol/kg of the amino variant had an increasedreduction of body weight over the body weight reduction of its pair fedgroup. Over the study OXM and liraglutide both significantly reducedbody weight, by 10.3% and 8.3% respectively. Measurement of glycemicprofile which monitored non-fasting glucose on days 1, 5, 12, 19, 26 and29 and fasting glucose on days 2, 9, 16, 23 and 30 had shown significantimprovement of these parameters, especially for the 6000 nmol/kg (FIG.9a, 9b ). Oral glucose tolerant test (OGTT) studies were performed ondays 2 and day 30 (FIGS. 10 and 11, respectively). The results showedthat MOD-6031 (the amino variant) significantly and dose-dependentlyimproved glucose tolerance with plasma glucose being significantlyreduced in the 1000, 3000 and 6000 nmoles/kg groups. Animals pair-fed tothe highest MOD-6031 dose exhibited a glucose excursion post glucosedose that was not significantly different to controls at any of the timepoints tested. On Day 2 of the OGTT studies, the improved glucoseprofile was associated with a delay of the insulin response, whichslightly delayed and gave higher stimulation for AUC 0-120 min (FIG.10). This can be due to inhibition of gastric emptying induced byMOD-6031's pharmacological activity which results in a delay in glucoserelease into the blood and a second insulin secretion phase. Day 30 ofthe OGTT studies was associated with a reduced insulin response comparedto controls showing that the compound improved insulin sensitivity (FIG.11). In addition, MOD-6031 dose-dependently reduced terminalcholesterol; the reduction observed with the 6000 nmoles/kg dose ofMOD-6031 was significantly greater than that of pair-fed counterparts(FIG. 12). All of these pharmacological improvements in body weight,food intake, glycemic and lipid profiles were greater not only thananimals treated bi-daily with OXM or liraglutide, but they were alsosignificantly greater than the effects observed in pair-fedcounterparts.

Terminal blood level of MOD-6031(PEG-FMS-OXM) and its hydrolyzedcompounds (PEG-FMS and OXM) were measured using an LC-MS/MS qualifiedmethod. Results showed dose dependent concentrations for the MOD-6031treated groups (Table 5). Comparison of this data to compound levels onday 2 (following single administration) showed that OXM peptide were notaccumulated during the study period when administered twice a week.PEG-FMS and PEG-FMS-OXM showed moderate accumulation over the study(Table 5). The actual concentration of MOD-6031 and OXM peptide for thetop dose of MOD-6031 at 24 h post last injection (Day 33) were 490 μg/mland 0.37 μg/ml, respectively. All samples from control animals werebelow the lower limit of the assay.

TABLE 5 Comparison of Plasma Concentrations 24 Hours Following SingleDose (Day 2) and Last Injection of Repeat MOD-6031 Dosing Regimen (Day33). compound: Day 2 Day 33 Increased by Dose: 1000 PEG-FMS-OXM 51.5767.51 1.31 3000 PEG-FMS-OXM 183.33 266.75 1.46 6000 PEG-FMS-OXM 296.33493.60 1.67 1000 OXM 0.07 0.09 1.29 3000 OXM 0.23 0.23 1.00 6000 OXM0.38 0.37 0.97 Dose*: 1000 PEG-FMS 65.73 78.04 1.19 3000 PEG-FMS 211.67295.75 1.40 6000 PEG-FMS 359.33 740.00 2.06 *Doses including impuritiesare 1515, 4545, and 9090 nmol/kg

Example 6 Improvement of Pharmacokinetic Parameters by MOD-6031 Variantin Ob/Ob Mice Model

Three groups (n=12) of ob/ob mice were singly administered with 1000,3000 and 6000 nmol/kg of MOD-6031 and were bled at 4, 8, 24, 36, 48, 72,96 and 120 h post administration (n=3 per time point) for PK analysisand the quantity of MOD-6031 and its compounds concentrations determinedLC-MS/MS method. Pharmacokinetic parameters such as Cmax, Tmax, AUC,T1/2Cl and Vz were calculated for MOD-6031 (PEG-FMS-OXM) and itshydrolyzed products; PEG-FMS and OXM, these parameters are presented inTable 6a, 6b and 6c, respectively. AUC 0-∞ was within 15% of AUC 0-t forall components at all doses, indicating that the sampling schedule wasadequate to characterize the pharmacokinetic profile of each component.For all three components, exposure appeared to be dose-proportional. Ingeneral, Cmax and AUC 0-t increased with dose and in approximately thesame proportion as the increase in dose.

Parameters for each component are expressed in molar concentrations inTable 7. Cmax values were approximately equivalent for PEG-FMS-OXM andPEG-FMS and lower for OXM. The observed T_(1/2) for PEG-FMS-OXM and OXMwere approximately 9 and 12 hours, respectively. The terminal T_(1/2)for PEG-FMS was much longer, approximately 30 hours. All samples fromcontrol animals and all samples collected prior to dosing were below thelower limit of the assay.

The pharmacokinetic and pharmacological data confirm the long actingproperties of MOD-6031. Twice a week dose of 3000 nmoles/kg of MOD-6031significantly reduced body weight and food consumption which wascomparable to twice a day of the OXM peptide treatment arm administeredat a 6000 nmoles/kg dose leading also to a significant reduction in drugload.

TABLE 6a PEG-FMS-OXM Pharmacokinetic Parameters Following SC Injectionof 1000, 3000, or 6000 nmoles/kg 1000 3000 nmol/kg, nmol/kg, 6000nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210 mg/kg Cmax μg/mL 70.2224 311 Tmax hr 8.00 8.00 8.00 AUC_(0-t) hr * μg/mL 1840 6330 10700AUC_(0-∞) hr * μg/mL 1850 6330 10700 T_(1/2) hr 8.57 8.80 12.3 CL/FmL/hr/kg 18.9 16.5 19.5 Vz/F mL/kg 234 210 346 Cmax/D (μg/mL)/ 2.01 2.141.48 (mg/kg) AUC_(1-∞)/D (hr * μg/mL)/ 52.9 60.5 51.3 (mg/kg)

TABLE 6b PEG-FMS Pharmacokinetic Parameters Following SC Injection of1000, 3000, or 6000 nmoles/kg of MOD-6031 1000 3000 nmol/kg, nmol/kg,6000 nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210 mg/kg Cmax pg/mL65.7 212 407 Tmax hr 24.0 24.0 36.0 AUC_(0-t) hr * μg/mL 3060 1070022800 AUC_(0-∞) hr * μg/mL 3280 11200 25800 T_(1/2) hr 33.5 22.8 35.0CL/F mL/hr/kg 14.0 12.4 10.8 Vz/F mL/kg 678 408 544 Cmax/D (μg/mL)/ 1.431.52 1.46 (mg/kg) AUC_(1-∞)/D (hr * μg/mL)/ 71.3 80.5 92.8 (mg/kg)

Note: Due to PEG-FMS impurity in the dosing solutions, the administereddoses of PEG-FMS (MOD-6031 plus PEG-FMS impurity) were 1515, 4545, and9090 nmol/kg instead of 1000, 3000 and 6000 nmol/kg, respectively.

TABLE 6c OXM Pharmacokinetics Parameters Following SC Injection of 1000,3000, or 6000 nmoles/kg of MOD-6031 1000 3000 nmol/kg, nmol/kg, 6000nmol/kg, Parameter Units 34.9 mg/kg 105 mg/kg 210 mg/kg Cmax μg/ml 0.1590.365 0.749 Tmax hr 8.00 8.00 8.00 AUC_(0-t) hr * μg/mL 3.19 9.29 18.5AUC_(0-∞) hr * μg/mL NC 9.42 18.5 T_(1/2) hr NC 11.7 11.8 CL/F mL/hr/kgNC 1420 1440 Vz/F mL/kg NC 23900 24400 Cmax/D (μg/mL)/ 0.0357 0.02740.0280 (mg/kg) AUC_(1-∞)/D (hr * μg/mL)/ NC 0.705 0.694 (mg/kg) NC = dueto the shape of the concentration versus time profile, parameters couldnot be calculated

TABLE 7 Pharmacokinetic Parameters Comparing the Three Components on aMolar Basis C_(max)/D AUC_(0-t)/D Dose^(a) C_(max) (nmol/mL)/ AUC_(0-t)(hr*nmol/mL)/ T_(1/2) nmol/kg Component nmol/mL (μmol/kg) hr*nmol/mL(μmol/kg) Hr 1000 PEG-FMS- 2.01 2.01 52.6 52.6 8.57 OXM 1515 PEG-FMS^(a)2.16 1.43 100 66.0 33.5 1000 OXM 0.0357 0.0357 0.716 0.716 NC 3000PEG-FMS- 6.42 2.14 181 60.3 8.80 OXM 4545 PEG-FMS^(a) 6.96 1.53 353 77.722.8 3000 OXM 0.0821 0.0273 2.09 0.697 11.7 6000 PEG-FMS- 8.90 1.48 30751.2 12.3 OXM 9090 PEG-FMS^(a) 13.4 1.47 750 82.5 35.0 6000 OXM 0.1680.0280 4.15 0.692 11.8 ^(a)Doses of PEG-FMS accounts for impurities(MOD-6031 plus PEG-FMS impurity).

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. A conjugate consisting of an oxyntomodulin, apolyethylene glycol polymer (PEG polymer) and 9-fluorenylmethoxycarbonyl(Fmoc) or 2-sulfo-9-fluorenylmethoxycarbonyl (FMS), wherein said PEGpolymer is attached to the amino terminus of said oxyntomodulin or to alysine residue on position number twelve (Lys₁₂) of said oxyntomodulin,via Fmoc or FMS.
 2. The conjugate of claim 1, wherein said oxyntomodulinconsists of the amino acid sequence set forth in SEQ ID NO:
 1. 3. Theconjugate of claim 1, wherein said PEG polymer is a PEG polymer with asulfhydryl moiety.
 4. The conjugate of claim 1, wherein said PEG polymeris PEG30.
 5. A pharmaceutical composition comprising the conjugate ofclaim 1 and a pharmaceutical acceptable carrier.
 6. A method of inducingglucose tolerance in a subject in need thereof, or increasing insulinsensitivity in a subject in need thereof, or inducing glycemic controlin a subject in need thereof, or reducing insulin resistance in asubject in need thereof, the method comprising administering thepharmaceutical composition of claim
 5. 7. A method of reducing foodintake in a subject in need thereof, or reducing body weight in asubject in need thereof, or for increasing energy expenditure in asubject in need thereof, or improving the cholesterol levels in asubject in need thereof, the method comprising administering thepharmaceutical composition of claim 5.